U.S. patent number 7,491,540 [Application Number 11/055,472] was granted by the patent office on 2009-02-17 for assay buffer, compositions containing the same, and methods of using the same.
This patent grant is currently assigned to Meso Scale Technologies, LLC. Invention is credited to Selen Altunata, Mark A. Billadeau, Eli N. Glezer, Larry Helms, Jonathan K. Leland, Svetlana Leytner, Mark Martin, George Sigal, Michael Tsionsky.
United States Patent |
7,491,540 |
Tsionsky , et al. |
February 17, 2009 |
Assay buffer, compositions containing the same, and methods of
using the same
Abstract
Compositions, reagents, kits, systems, system components, and
methods for performing assays. More particularly, the invention
relates to the use of novel combinations of reagents to provide
improved assay performance.
Inventors: |
Tsionsky; Michael
(Gaithersburg, MD), Glezer; Eli N. (Chevy Chase, MD),
Altunata; Selen (Rockville, MD), Sigal; George
(Rockville, MD), Leland; Jonathan K. (Silver Spring, MD),
Billadeau; Mark A. (Knoxville, MD), Leytner; Svetlana
(Austin, TX), Martin; Mark (Rockville, MD), Helms;
Larry (Germantown, MD) |
Assignee: |
Meso Scale Technologies, LLC
(Gaithersburg, MD)
|
Family
ID: |
26981405 |
Appl.
No.: |
11/055,472 |
Filed: |
February 9, 2005 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20050136497 A1 |
Jun 23, 2005 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10238437 |
Sep 10, 2002 |
6919173 |
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60363498 |
Mar 11, 2002 |
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60318289 |
Sep 10, 2001 |
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Current U.S.
Class: |
436/8; 436/18;
435/968; 435/6.12 |
Current CPC
Class: |
G01N
21/76 (20130101); G01N 33/5306 (20130101); G01N
33/573 (20130101); C12Q 1/485 (20130101); G01N
33/582 (20130101); G01N 33/58 (20130101); G01N
33/54386 (20130101); Y10T 436/10 (20150115); G01N
2458/30 (20130101); Y10T 436/108331 (20150115); Y10S
435/968 (20130101); Y10S 435/967 (20130101); G01N
2458/40 (20130101); G01N 2333/91205 (20130101) |
Current International
Class: |
G01N
31/00 (20060101); C12Q 1/68 (20060101) |
Field of
Search: |
;435/6,968
;436/18,8 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
Primary Examiner: Riley; Jezia
Attorney, Agent or Firm: Scully, Scott, Murphy and Presser,
P.C.
Parent Case Text
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of copending application U.S. Ser.
No. 10/238,437, filed Sep. 10, 2002, which claims priority to U.S.
Provisional Application Ser. No. 60/318,289, filed Sep. 10, 2001,
and U.S. Provisional Application Ser. No. 60/363,498, filed Mar.
11, 2002, each of which are hereby incorporated by reference.
Claims
The invention claimed is:
1. An aqueous reagent composition consisting essentially of: (a)
water; (b) piperazine-N,N'-bis(2-ethanesulfonic acid) at a
concentration between about 10 mM and about 200 mM; (c) an
electrolyte comprising a pH buffer selected from the group
consisting of tris(hydroxymethyl)aminomethane, glycylglycine,
phosphate and combinations thereof, wherein the concentration of
the pH buffer is between about 40 mM and about 1000 mM; and (d) a
phenyl ether-containing surfactant; wherein said reagent
composition has a pH between about 6.5 and about 8.5.
2. An aqueous reagent composition consisting of: (a) water; (b)
piperazine-N,N'bis(2-ethanesulfonic acid) at a concentration
between about 10 mM and about 200 mM; (c) an electrolyte comprising
a pH buffer selected from the group consisting of
tris(hydroxymethyl)aminomethane, glycylglycine, phosphate and
combinations thereof wherein the concentration of the ph buffer is
between about 40 mM and about 1000 mM; and (d) a phenyl
ether-containing surfactant; wherein said reagent composition has a
ph between about 6.5 and about 8.5.
3. The aqueous reagent composition of claim 1, wherein said
electrolyte further comprises a component selected from the group
consisting of potassium ions, chloride ions, and mixtures
thereof.
4. The aqueous reagent composition of claim 2, wherein said
electrolyte further comprises a component selected from the group
consisting of potassium ions, chloride ions, and mixtures
thereof.
5. The aqueous reagent composition of claim 1, wherein (a) said pH
buffer is phosphate at a concentration less than 400 mM; and (b)
said surfactant is at a concentration between 0.05% and 0.5%.
6. The aqueous reagent composition of claim 2, wherein (a) said pH
buffer is phosphate at a concentration less than 400 mM; and (b)
said surfactant is at a concentration between 0.05% and 0.5%.
7. The aqueous reagent composition of claim 5, wherein said
surfactant is selected from the group consisting of Triton X-100,
NP-40 and mixtures thereof.
8. The aqueous reagent composition of claim 7, wherein said
surfactant is selected from the group consisting of Triton X-100,
NP-40 and mixtures thereof.
9. The aqueous reagent composition of claim 1 further comprising a
preservative.
10. An aqueous reagent composition consisting of: (a) water: (b)
piperazine-N,N'-bis(2-ethanesulfonic acid) at a concentration
between about 10 mM and about 200 mM; (c) an electrolyte comprising
a pH buffer selected from the group consisting of
tris(hydroxymethyl)aminomethane, glycyciglycine, phosphate and
combinations thereof, wherein the concentration of the pH buffer is
between about 40 mM and about 1000 mM; and (d) a phenly
ether-containing surfactant; and (e) a preservative; wherein said
reagent ent composition has a pH between about 6.5 and about
8.5.
11. The aqueous reagent composition of claim 1 comprising 10-200 mM
PIPES, 40-1000 mM potassium phosphate and 0.2%-2% Triton X-100, pH
7.5.+-.0.05.
12. The aqueous reagent composition of claim 2 comprising 10-200 mM
PIPES, 40-1000 mM potassium phosphate and 0.2%-2% Triton X-100, pH
7.5%.+-.0.05.
13. The aqueous reagent composition of claim 1 comprising 20-50 mM
PIPES, 75-150 mM potassium phosphate and 0.05%-0.5% Triton X-100,
pH 7.5.+-.0.05.
14. The aqueous reagent composition of claim 2 comprising 20-50 mM
PIPES, 75-150 mM potassium phosphate and 0.05%-0.5% Triton X-100,
pH 7.5.+-.0.05.
Description
1. FIELD OF THE INVENTION
This application relates to compositions for use in assays,
particularly in electrochemiluminescent assays, and methods of
using the same.
2. BACKGROUND OF THE INVENTION
At this time, there are a number of commercially available
instruments that utilize electrochemiluminescence (ECL) for
analytical measurements including drug screening. Species that can
be induced to emit ECL (ECL-active species) have been used as ECL
labels. Examples of ECL labels include: i) organometallic compounds
where the metal is from, for example, the noble metals of group
VIII, including Ru-containing and Os-containing organometallic
compounds such as the tris-bipyridyl-ruthenium (RuBpy) moiety and
ii) luminol and related compounds. Species that participate with
the ECL label in the ECL process are referred to herein as ECL
coreactants. Commonly used coreactants include tertiary amines
(e.g., see U.S. Pat. No. 5,846,485, herein incorporated by
reference), oxalate, and persulfate for ECL from RuBpy and hydrogen
peroxide for ECL from luminol (see, e.g., U.S. Pat. No. 5,240,863).
The light generated by ECL labels can be used as a reporter signal
in diagnostic procedures (Bard et al., U.S. Pat. No. 5,238,808).
For instance, an ECL label can be covalently coupled to a binding
agent such as an antibody, nucleic acid probe, receptor or ligand;
the participation of the binding reagent in a binding interaction
can be monitored by measuring ECL emitted from the ECL label.
Alternatively, the ECL signal from an ECL-active compound may be
indicative of the chemical environment (see, e.g., U.S. Pat. No.
5,641,623 which describes ECL assays that monitor the formation or
destruction of ECL coreactants). For more background on ECL, ECL
labels, ECL assays and instrumentation for conducting ECL assays
see U.S. Pat. Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581;
5,597,910; 5,641,623; 5,643,713; 5,679,519; 5,705,402; 5,846,485;
5,866,434; 5,786,141; 5,731,147; 6,066,448; 6,136,268; 5,776,672;
5,308,754; 5,240,863; 6,207,369 and 5,589,136 and Published PCT
Nos. WO99/63347; WO00/03233; WO99/58962; WO99/32662; WO99/14599;
WO98/12539; WO97/36931 and WO98/57154.
Commercially available ECL instruments have demonstrated
exceptional performance. They have become widely used for reasons
including their excellent sensitivity, dynamic range, precision,
and tolerance of complex sample matrices. The commercially
available instrumentation uses flow cell-based designs with
permanent reusable flow cells. Recently, ECL instrumentation has
been disclosed that uses reagents immobilized on the electrode used
to induce ECL (see, e.g., U.S. Pat. Nos. 6,140,045; 6,066,448;
6,090,545; 6,207,369 and Published PCT Application No. WO98/12539).
Multi-well plates having integrated electrodes suitable for such
ECL measurements have also been recently disclosed (see, e.g., U.S.
application Ser. Nos. 10/185,274 and 10/185,363, entitled "Assay
Plates, Reader Systems and Methods for Luminescence Test
Measurements", each filed on Jun. 28, 2002 and hereby incorporated
by reference). See also, U.S. application Ser. No. 60/318,293 ,
(Entitled: "Methods and Apparatus for Conducting Multiple
Measurements on a Sample" by Glezer et al.), filed on even date
herewith, hereby incorporated by reference.
Currently, pH buffers containing inorganic phosphate are employed
in many electrochemiluminescence assays. Applicants have discovered
that such pH buffers can, in certain assays, interfere with the
assay and decrease the performance of the assay.
Accordingly, it would be desirable to find alternative pH assay
buffers, compositions containing the same and methods of using the
same for use in those assays which are detrimentally effected by pH
buffers containing inorganic phosphate. It would also be desirable
to find alternative ECL Assay Buffers with improved performance in
ECL assays.
3. SUMMARY OF THE INVENTION
The present invention relates to improved compositions, reagents,
kits, systems, system components, and methods for performing
assays. More particularly, the invention relates to the use of
novel combinations of reagents to provide improved assay
performance.
One aspect of the invention relates to improved ECL Assay Buffers
that comprise an ECL coreactant and, preferably, a pH buffering
agent. The ECL Assay Buffers provide a suitable environment for
efficiently inducing ECL labels to emit ECL and for sensitively
measuring ECL labels via the measurement of ECL. The ECL Assay
Buffers of the invention may optionally comprise additional
components including detergents, preservatives, anti-foaming
agents, ECL active species, salts, metal ions and/or metal
chelating agents. The ECL Assay Buffers of the invention may also
include components of a biological assay, which in some cases may
be labeled with an ECL label, including binding reagents, enzymes,
enzyme substrates, cofactors and/or enzyme inhibitors. The
invention also includes assay reagents, compositions, kits, systems
and system components that comprise the ECL Assay Buffers of the
invention and, optionally, additional assay components. The
invention also includes methods for conducting ECL assays using the
ECL Assay Buffers of the invention.
Another aspect of the invention relates to the use of pH buffers
which are substantially free of inorganic phosphates. Such buffers,
in some applications, have been found to significantly improve the
performance of ECL measurements. Such buffers have also been found
to be advantageous in certain applications where phosphate has been
found to interfere with a chemical, biochemical or biological
reaction.
Surprisingly, such reagents provide a number of surprising
advantages including improving the performance of assays employing
phospho-specific antibodies (i.e., antibodies that specifically
bind with a phospho-peptide, phospho-amino acid and/or
phospho-protein). It is believed that these antibodies may have a
low affinity for inorganic phosphate and that the elimination of
the inorganic phosphate greatly reduces interference between the
phosphate of the pH buffer and the phospho-specific antibodies.
Accordingly, the invention includes method, reagents, kits and
compositions for measuring phospho-peptides, phospho-amino acids or
phospho-protein which use buffer compositions that are free or
substantially free (e.g., below the levels that interfere with
phospho-specific antibodies). Such methods, kits, compositions, and
reagents are, preferably, applied to the measurement (most
preferably using ECL detection) of protein kinase or phosphorylase
activities through the specific measurement of reaction products or
substrates.
Another aspect of the invention relates to compositions and
reagents with that give high signal to background ratios in
electrochemiluminescence assays. Such improved performance has been
achieved through the identification of advantageous combinations of
ECL coreactants, pH buffers, detergent and pH and, in particular,
through the use of ECL coreactants and/or pH buffers other than TPA
and phosphate. These improved formulations are of particular value
in non-wash assays and high sensitivity assays. In some embodiments
of the invention, the performance of ECL assays is improved even
further through optimal combinations of reagent compositions with
electrode compositions.
In some embodiments of the invention, the compositions and reagents
of the invention improve the ratio of ECL signal from bound label
to ECL signal from free label. This is particularly true in assays
involving reagents immobilized on a solid surface such as an
electrode. This is important, for example, in solid phase assays
not having a wash step (especially in low affinity interaction
assays) since the major component of the background signal comes
from the labels present in solution.
Yet another advantage of the invention relates to improved
sensitivity of assays using the compositions of the invention. More
specifically, the ECL Assay Buffers of the invention provide
improved sensitivity at low detection levels by reducing the
background electrochemiluminescence in the absence of ECL labels.
Surprisingly, ECL Assay Buffers comprising pH buffering agents
other than phosphate or which are substantially free of inorganic
phosphate emit less background luminescence than conventional ECL
Assay Buffers comprising inorganic phosphate based pH buffers. This
is particularly advantages at low detection levels where increasing
the signal to background ratio greatly improves the performance of
the assay.
Another aspect of the invention relates to improved reagent kits
comprising the ECL assay buffers, where the reagents include
non-phosphate based pH buffering agents, the ECL assay buffers are
substantially free of inorganic phosphate and/or the ECL assay
buffers employ tertiary amine coreactants other than TPA. In
particular, kits containing, in one or more containers, the ECL
assay buffer and, preferably also containing one or more other
assay components.
Another aspect of the invention relates to improved methods
performed using the present invention, particularly assay methods
employing phospho-specific antibodies, low detection limits,
immobilized reagents and/or a non-wash formats.
Yet another aspect of the invention relates to improved systems and
apparatus containing the compositions or reagents of the invention
and/or improved systems and apparatus adapted to perform the
improved methods of the invention.
4. DESCRIPTION OF THE FIGURES
FIG. 1 compares the rates of dissociation of a
phosphopeptide-antiphosphopeptide complex in three different ECL
Assay Buffers that comprise different pH buffering agents.
FIG. 2 shows the rate of dissociation of a
phosphopeptide-antiphosphopeptide complex in an ECL Assay Buffer
that comprises TPA as an ECL coreactant and Tris as a pH buffering
agent. The complex was not washed to remove free antibody prior to
addition of the ECL Assay Buffer.
FIG. 3 is a graphical representation of an end-product stability
study comparing the dissociation rate of an anti-phosphotyrosine
antibody from autophosphorylated EGF receptor in two different ECL
Assay Buffers: 150 mM TPA/150 mM Phosphate and 100 mM TPA/400 mM
glycylglycine. The concentration of the labeled
.alpha.-phosphotyrosine antibody was 6.7 nM.
FIG. 4 compares the performance of four different ECL Assay Buffers
in the ECL measurement of a labeled reagent that was immobilized on
the surface of an unetched (FIG. 4A) or plasma etched (FIG. 4B)
carbon ink electrode. The figure shows the signals from surface
bound reagent, the background signal measured in the absence of the
bound reagent and the signal to background ratio (S/B).
FIG. 5 compares the performance of four different ECL Assay Buffers
in the ECL measurement of a labeled reagent that was immobilized on
the surface of an unetched (FIG. 5A) or plasma etched (FIG. 5B)
carbon ink electrode. The figure shows the signals from surface
bound reagent, the background signal measured in the absence of the
bound reagent and the signal to background ratio (S/B). The figure
also shows the signal obtained when a non-surface bound labeled
reagent was introduced into the ECL Assay Buffers and the ratio of
the signals from the surface bound and non-surface bound reagents
(B/F).
FIG. 6 compares the effect of three different detergents on the ECL
signal from a labeled reagent that was immobilized on the surface
of a plasma etched carbon ink electrode. The detergents were
introduced into an ECL Assay Buffer comprising TPA and phosphate
(FIG. 6A) or PIPES and phosphate (FIG. 6B). The figure shows the
signals from surface bound reagent, the background signal measured
in the absence of the bound reagent and the signal to background
ratio (S/B).
FIG. 7 compares the effect of five different detergents on the ECL
signal from a labeled reagent that was immobilized on the surface
of a non-etched carbon ink electrode. The detergents were
introduced into four different ECL Assay Buffers differing in the
identity of the ECL coreactant or pH buffering agent. The figure
shows the signals from surface bound reagent, the background signal
measured in the absence of the bound reagent and the signal to
background ratio (S/B).
5. DETAILED DESCRIPTION OF THE INVENTION
The invention, as well as additional objects, features and
advantages thereof, will be understood more fully from the
following detailed description of certain preferred
embodiments.
An ECL-active species may be referred to as an ECL moiety, ECL
label, ECL label compound or ECL label substance, etc. It is within
the scope of the invention for these ECL-active species--when
utilized in certain of the composition, reagent, kit, method, or
system embodiments in accordance with the invention--to be linked
to other molecules and, in particular, to components of biochemical
or biological assays, e.g., an analyte or an analog thereof, a
binding partner of the analyte or an analog thereof, a further
binding partner of such aforementioned binding partner, or a
reactive component capable of binding with the analyte, an analog
thereof or a binding partner as mentioned above. The
above-mentioned species can also be linked to a combination of one
or more binding partners and/or one or more reactive components. In
certain enzymatic assays, an ECL-active species may be linked to an
enzyme substrate.
It is similarly within the scope of the invention for the
aforementioned "composition", hereinafter sometimes an "ECL,
composition", or a "system" to contain unstable, metastable and
other intermediate species formed in the course of the ECL
reaction, such as an ECL moiety in an excited state as aforesaid
and the above-mentioned strong reducing agent. Additionally,
although the emission of visible light is an advantageous feature
of certain embodiments of the invention it is within the scope of
the invention for the composition (hereinafter sometimes "ECL
composition") or system to emit other types of electromagnetic
radiation, such as infrared or ultraviolet light, X-rays,
microwaves, etc. Use of the terms "electrochemiluminescence",
"electrochemiluminescent", "electrochemiluminesce", "luminescence",
"luminescent" and "luminesce" in connection with the present
invention does not require that the emission be light, but admits
of the emission's being such other forms of electromagnetic
radiation.
The present invention relates to ECL assay buffers, assay
compositions containing the same, and methods of using the same. As
stated above, several disadvantages were discovered when using the
phosphate based ECL assay buffer of the prior art. More
specifically, it was found during the development of a specific ECL
assay for tyrosine kinase activity that the phosphate in a standard
formulation of the ECL coreactant TPA (ORIGEN Assay Buffer, IGEN
International: 200 mM Phosphate, .about.100 mM TPA, pH .about.7.5)
disrupted the binding between an phospho-specific antibody and a
phosphorylated substrate. The assay involved i) the
kinase-dependent phosphorylation of a peptide immobilized on a
carbon electrode; ii) the specific binding of a labeled (with a
derivative of Ru(bpy).sub.3) phospho-specific antibody; iii) the
addition of the ECL coreactant tripropylamine (TPA) and iv) the
detection of ECL from the bound label (see, e.g., Example A below).
Applicants discovered that when TPA was added by the addition of
ORIGEN Assay Buffer that the measured ECL signal was sharply
dependent on the time the binding complex was left exposed to the
ORIGEN Assay Buffer (FIG. 1); the measured signal dropped sharply
over time. In fact, after a 1-hour incubation only a small fraction
(.about.10%) of initial signal was detected. The affinity of the
pY20 antibody (Zymed Lab) used in the assay toward the
phospho-tyrosine sites that were formed at the surface of the plate
during the enzymatic reaction was much greater than toward free
phosphate in solution. However, the high concentration of free
phosphate in ORIGEN assay buffer (200 mM) is now believed to have
caused the dissociation of phospho-tyrosine/pY20 complex, resulting
in the signal decaying sharply.
One way around this problem was to have a fixed time between the
dispensing of ECL assay buffer and the read step so that the signal
decay is calibrated and subtracted. However, this approach is not
desirable in high throughput screening applications, where
robustness of the assay and flexibility of dispensing protocol are
desired.
Thus, a number of different organic pH buffers were tested as
alternatives to the conventional phosphate based assay buffer. Many
of the conventional biological buffers (including Tricine, HEPES,
MOPS, BES--all from Sigma), however, interfered with the ECL
generation from TPA and provided only 2-20 % of ECL signal observed
with the ORIGEN assay buffer. Applicants, however, discovered a set
of buffers that provided ECL signals that were comparable to the
signal observed in TPA/phosphate.
Accordingly, applicants have discovered that substitution of the
phosphate buffer with a pH buffer which was substantially free of
inorganic phosphate can ECL signal comparable to the signal
observed in standard ORIGEN assay buffer, without the
above-described disadvantages. Preferably, the pH buffer is free of
inorganic buffer.
Furthermore, applicants have discovered that the phosphate-free ECL
assay buffers of the invention are not only beneficial when applied
to phosphopeptide binding assays but have other beneficial
properties (including lower background signals) that may improve a
wide range of ECL assays.
Furthermnore, applicants have discovered ECL assay buffer
background reducing agents that, when introduced into ECL assay
buffers reduce ECL assay buffer background and improve assay
performance. These agents are, preferably, also pH buffering
agents, most preferably, GlyGly or Tris.
Furthermore, applicants have discovered novel ECL assay buffers
that employ ECL coreactants other than the traditional TPA.
Surprisingly, a number of coreactants have been discovered to
generate ECL signals that are comparable to those generated with
TPA. In addition, the use of ECL coreactants other than TPA have
been found to improve the performance of non-washed ECL assays
through their improved ability, relative to TPA, to discriminate
between ECL labels that are held in proximity to an electrode and
labels that are free in solution. The use of coreactants other than
TPA has additional benefits due to the higher water solubility and
lower vapor pressure of some of the new coreactants that have been
identified.
Furthermore, applicants have discovered that the presence or
absence of detergents can have profound impact on the performance
of an ECL assay buffer. Surprisingly, the effect of detergents on
ECL can be influenced by the choice of ECL coreactant and working
electrode material. Applicants have developed detergent-containing
ECL assay buffers suitable for a variety of different applications
and ECL systems.
As noted above, one aspect of the invention relates to improved ECL
Assay Buffers that comprise an ECL coreactant and, preferably, a pH
buffering agent. The ECL Assay Buffers provide a suitable
environment for efficiently inducing ECL labels to emit ECL and for
sensitively measuring ECL labels via the measurement of ECL. The
ECL Assay Buffers of the invention may optionally comprise
additional components including detergents, preservatives,
anti-foaming agents, ECL active species, salts, metal ions and/or
metal chelating agents. The ECL Assay Buffers of the invention may
also include components of a biological assay, which in some cases
may be labeled with an ECL label, including binding reagents,
enzymes, enzyme substrates, cofactors and/or enzyme inhibitors.
Preferably, the ECL assay buffers of the invention are aqueous or
substantially aqueous in nature, although it may be desirable in
some applications to add organic cosolvents such as DMSO, DMF,
methanol, ethanol or other alcohols. In one embodiment of the
invention, an ECL assay buffer (or one or more components thereof)
is provided in dry form and the user forms the ECL assay buffer
solution by addition of the appropriate solvent or matrix
(preferably a water or an aqueous medium).
5.1 ECL Coreactants
Most, if not all, current commercial applications of ECL technology
involve the measurement of ECL labels (and, in particular,
organometallic complexes of ruthenium) in the presence of an ECL
assay buffer containing tri-n-propylamine (TPA) as a coreactant and
phosphate as a pH buffering agent. These ECL assay buffers have
been optimized for and have provided excellent performance in
commercial ECL instrumentation that employ, as a solid phase for
binding assays, magnetic particles that are collected on the
surface of a metal (typically, platinum) electrode.
Applicants have discovered that in some applications, certain
functionalized tertiary alkylamines can provide performance that is
comparable or better to TPA. These functionalized tertiary amines
are especially useful in assays employing carbon-based electrodes
(e.g., electrodes comprising carbon particle or carbon nanotubes
including composite materials such as plastics and inks) and/or
assay reagents (such as binding reagents) that are immobilized onto
electrodes. The functionalized tertiary alkylamines of the
invention, preferably, have one or more of the following
properties: i) they are oxidized on carbon-based electrodes in a
one electrode oxidation to give an amine radical cation which can
subsequently lose a proton to form a radical reductant (Scheme 1);
ii) they have an oxidation potential on carbon-based electrodes
that is comparable (within 150 mV) or greater than that of
Ru(II)(bpy).sub.3; iii) they can be oxidized, most preferably at a
pH between 6 and 9, at a potential less than that required to
breakdown water at a carbon-based electrode; iv) the energy
released by the reaction of the radical reductant with
Ru(III)(bpy)3 to produce Ru(II)(bpy)3 is sufficient to produce
Ru(II)(bpy)3 in a luminescent excited state and v) the lifetimes of
the amine radical cation and/or radical reductant are less than the
corresponding TPA-derived species.
##STR00001##
Applicants have discovered that, through the use of the
ftnctionalized tertiary alkylamines of the invention, it is
possible to improve the selectivity of ECL excitation at an
electrode for ECL labels bound to the electrode (as opposed to ECL
labels that are free in solution). Without being bound by theory,
it is believed that this increased selectivity is due to the lower
lifetimes of the amine radical cation and/or radical reductant
relative to the corresponding TPA-derived species (thus limiting
the participation of the reactive species to ECL reactions that
occur proximate to the electrode surface). Preferably, the
diffusion distance of the amine radical cation and/or radical
reductant (the distance that the species can diffuse during its
lifetime) is less than 1 .mu.m, more preferably, <500 nm, even
more preferably less than 100 nm, even more preferably less than 50
nm and most preferably <10 nm. The high selectivity between free
and bound labels has led to improved sensitivity in non-washed ECL
assay formats. The ratio of signal from bound label and free label
(B/F ratio) may improved by replacing TPA with a non-TPA coreactant
of the invention. This improvement is preferably greater than a
factor of 2, more preferably greater than a factor of 5 and most
preferably greater than a factor of 10.
The functionalized tertiary amine coreactants of the invention,
preferably have the structure NR.sup.1R.sup.2R.sup.3, wherein
R.sup.1, R.sup.2 and R.sup.3 are alkyl groups comprising at least
2, preferably 3, carbons and wherein one or more of R.sup.1,
R.sup.2 and R.sup.3 are functionalized with a hydrophilic
functional group, more preferably a charged group, most preferably
a negatively charge group. Preferred functional groups include
hydroxyl, dialkylamino, sulfate, sulfonate, carboxylate and
carboxylic acid ester.
Especially preferred coreactants include compounds with the
structure (n-Pr).sub.2N(CH.sub.2).sub.nR, wherein n is greater than
or equal to 2 (more preferably 3), and R is a hydrophilic
flunctional group as defined above, preferably, carboxylate,
dialkylamino (more preferably dipropylamino) or most preferably
sulfonate.
Other preferred coreactants include compounds with the
structure
##STR00002##
Wherein i) X is --(CH.sub.2)--or a heteroatom, preferably --O--,
--S--, or --N(R.sup.1)--; ii) R and R.sup.1 are alkyl groups
comprising 2 or more (preferably 3 or more) carbons; iii) n and m
are each greater than or equal to 1 and are preferably two and iv)
R (and, optionally R.sup.1) comprise a hydrophilic functional group
as defined above. Most preferably, R is
--(CH.sub.2).sub.n--F.sup.1, wherein n is greater than or equal to
3 and F.sup.1 is a hydrophilic functional group, preferably,
carboxylate or sulfonate. In the cases where X is --N(R.sup.1)--,
R.sup.1 is, most preferably, --(CH.sub.2).sub.n--F.sup.2,wherein n
is greater than or equal to 3 and F.sup.1 is H, alkyl, or a
hydrophilic fuinctional group, most preferably, carboxylate or
sulfonate.
Many of the so-called "Good" buffers (Good et al., Biochemistry, 5,
467 (1966); Good et al., Methods in Enzymol., 24, Part B, 53 (1972)
and Ferguson et al., Anal. Biochem., 104, 300 (1980)) have tertiary
amnines and have been found to act as ECL coreactants on carbon
electrodes. These "Good" buffers, generally have tertiary amines
having piperazine or morpholine cores. Specific amines that act as
ECL coreactants on carbon-based electrodes include:
3-(di-n-propylamino)-propanesulfonic acid;
4-(di-ni-propylamino)-butanesulfonic acid;
4-[bis-(2-hydroxyethane)-amino]-butanesulfonic acid;
piperidine-N-(3-propanesulfonic acid); azepane-N-(3-propanesulfonic
acid); piperidine-N-(3-propionic acid) (PPA);
3-morpholino-2-hydroxypropanesulfonic acid (MOPSO);
3-morpholinepropanesulfonic acid (MOPS); N-(2-hydroxyethyl)
piperazine-N'-3-propanesulfonic acid (EPPS);
N-(2-hydroxyethyl)piperazine-N'-3-ethanesulfonic acid (BES);
piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES);
triethanolamine; N-2-hydroxypiperazine-N-2-ethanesulfonic acid
(HEPES); piperazine-N,N'-bis-4-butanesulfonic acid;
homopiperidine-N-3-propanesulfonic acid;
piperazine-N,N'-bis-3-propanesulfonic acid;
piperidine-N-3-propanesulfonic acid;
piperazine-N-2-hydroxyethane-N'-3-methylpropanoate;
piperazine-N,N'-bis-3-methylpropanoate;
1,6-diaminohexane-N,N,N',N'-tetraacetic acid; N,N-bis
propyl-N-4-aminobutanesulfonic acid;
N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES);
1,3-bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane);
3-dimethylamino-1-propanol; 3-dimethylamino-2-propanol;
N,N,N',N'-tetrapropylpropane-1,3,-diamine (TPA dimer);
piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO) and
2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic
acid (HEPPSO). HEPES. POPSO, HEPPSO, EPPSO, PPA and PIPES are
especially preferred for their high signals and high discrimination
between bound and free labels. TES is also preferred for its high
signal.
Additional coreactants include proline, peptides having an
N-terminal proline. Preferably, the proline is N-alkylated to form
a tertiary amine.
The use of coreactants having hydrophilic functional groups (and,
in particular, coreactants that are zwitterionic at neutral pH) has
a variety of advantages that are unrelated to their ability to act
as ECL coreactants. These species tend to be highly water soluble
and to have low vapor pressure. For these reasons it is possible to
produce highly concentrated stock solutions that may be diluted as
necessary for use. It is also possible prepare dried reagents
comprising the coreactants without uncertainty due to loss of
coreactant in the vapor phase. Furthermore, when present in dried
reagents, these coreactants resolubilize quickly in a minimum of
volume.
5.2 pH Buffering Agents
Conventional ECL assay buffers optimized for use with commercial
ECL instruments have typically comprised TPA in a phosphate-based
pH buffer. These formulations have been especially useful for
conducting solid phase assays employing magnetic particles that are
captured on an electrode. Applicants have discovered that in some
applications, other pH buffering agents (including organic pH
buffers) can provide performance that is comparable or better to
phosphate. These non-phosphate pH buffers (and pH buffer solutions
and ECL assay buffers comprising these buffers are especially
useful in assays employing carbon-based electrodes (e.g.,
electrodes comprising carbon particle or carbon nanotubes including
composite materials such as plastics and inks) and/or assay
reagents (such as binding reagents) that are immobilized onto
electrodes. They are also advantageous for use in assays where
phosphate is an interferent. Preferably, ECL assay buffers
employing the non-phosphate buffers of the invention have less than
15 mM inorganic phosphate, more preferably-they have less than 5 mM
inorganic phosphate, even more preferably they have less than 1 mM
phosphate, even more preferably they are substantially free of
inorganic phosphate, most preferably they are free of inorganic
phosphate.
The pH buffering agent, preferably, is not oxidized under the
conditions used to generate ECL and do not interfere with the
generation of ECL. Two pH buffers that have proved especially
useful are tris-(hydroxymethyl)aminomethane (Tris) and
oligo(glycines), preferably glycyl-glycine (Gly-Gly). Applicants
have discovered that ECL assays on carbon-based electrodes using
TPA/Tris or TPA/Gly-Gly ECL assay buffers have comparable signals
from electrode-bound ECL labels as those observed with conventional
TPA/phosphate buffers. The background signals in the absence of ECL
labels, however, are considerably less with the Tris and Gly-Gly
buffers. This reduction in the background signal leads to an
increase in the ratio of signal to background (S/B) and an increase
in the sensitivity of ECL assays using the new formulations.
Preferably, the ECL assay buffers of the invention have S/B ratios
that are 2-fold, more preferably 5-fold and, most preferably,
10-fold better than those obtained using phosphate-based
systems.
Without being bound by theory, applicants hypothesize that the
improved performance of the Tris and Gly-gly based ECL assay
buffers is related to an ability of the buffering agents to act as
ECL assay buffer reducing agents by reacting with and destroying
tertiary amine oxidation products and/or other reactive oxidized
species (e.g., amine radical cations and radical reductants) that
are responsible for the assay buffer background. This effect is
most pronounced away from the electrode surface where the
concentration of these species are lower, so the Tris and Gly-gly
components do not affect signals from electrode-bound labels. The
Tris and Gly-gly buffers may also improve the observed bound to
free ratios, although this effect is less than that observed by
switching to non-TPA buffers such as PIPES.
Applicants have found that the Tris and Gly-gly buffering systems
are also suitable for use with non-TPA coreactants such as PIPES.
When using coreactants such as PIPES that may act as pH buffers, it
may be possible to omit additional buffering agents.
5.3 Detergents
Applicants have discovered that the presence or absence of
detergents can have a surprisingly large effect on ECL signals. The
nature of this effect is, unexpectedly, dependent on the electrode.
On oxidized electrodes (e.g., plasma-oxidized carbon inks or plasma
oxidized polymer composites containing carbon particles or carbon
nanotubes) exposed to TPA-containing ECL assay buffers; the effect
appears to be relatively small except in the case of phenyl ether
containing detergents such as the Triton and Nonidet series of
detergents (e.g., Triton X-100). A common method for generating ECL
is through the use of a ramp potential. In general a plot of ECL
intensity vs. applied potential has the form of a peak. ECL
increases as the oxidation potentials of the label and coreactant
are approached. On scanning past this potential, the ECL intensity
eventually begins to drop as the coreactant is consumed and water
oxidation begins to occur. Applicants have observed that the
addition of phenyl ether containing detergents leads to the
addition of a small ECL peak at higher potential than the main ECL
peak. This peak occurs at a potential similar to the an oxidation
wave observed with pure Triton X-100, thus leading applicants to
speculate that the new peak is associated with the oxidation of the
detergent (or an associated impurity) and the participation of the
oxidation products in an ECL reaction.
The behavior on non-oxidized carbon-based electrodes (and, in
particular, untreated carbon ink electrodes) is very different. On
these electrodes the ECL signal in the presence of TPA-containing
buffers (as well as the S/B ratio) is drastically improved by the
addition of detergent. This effect appears to be relatively
independent of the nature of the detergent (although non-ionic
detergents are preferred due to their relatively weak ability to
denature biological systems), but requires the concentration of
detergent to be roughly equal to or greater than the critical
micellar concentration (cmc) of the detergent. In preferred
embodiments of the invention, the addition of detergent to an ECL
assay buffer leads to an improvement in assay signal or S/B
(preferably induced with a carbon-based electrode, most preferably
a carbon-ink electrode) of greater than a factor of 2, more
preferably greater than a factor of 5 and most preferably greater
than a factor of 10.
The behavior of non-TPA containing ECL assay buffers and, in
particular, non-TPA containing ECL assay buffers (especially,
buffers comprising the non-TPA tertiary amine coreactants of the
invention, preferably comprising N-substituted morpholines or
piperazines, most preferably comprising PIPES) appears to be less
dependent on the nature of a carbon electrode. For example,
applicants have found that assays involving the use of PIPES as a
coreactant, on both oxidized and non-oxidized electrodes, are
unexpectedly and significantly improved by the addition of phenyl
ether containing substances, and, in particular, phenyl ether
containing detergents. Other detergents that did not possess the
phenyl ether moiety did not produce this effect. In preferred
embodiments of the invention, the addition of detergent to a
non-TPA based ECL assay buffer (preferably, a PIPES-based ECL assay
buffer) leads to an improvement in assay signal or S/B (preferably
induced with a carbon-based electrode, most preferably a carbon-ink
electrode) of greater than a factor of 10, more preferably greater
than a factor of 30 and most preferably greater than a factor of
100.
In certain assays, e.g., assays involving detergent sensitive
components such as biological membranes, it may be advantageous to
reduce (e.g., to <0.1%) or eliminate detergents from ECL Assay
Buffers. It should be understood that the various detergent
containing formulations of the invention may also be prepared in
low detergent or detergent-free forms for these detergent sensitive
applications. In preferred embodiments, assays employing detergent
sensitive components employ ECL Assay Buffers containing one of the
following coreactants: TPA,
N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid, or
A.sub.2N--(CH.sub.2).sub.n--NB.sub.2, where A and B are alkyl
groups (preferably, propyl) and n is an integer (preferably 3 or 4,
most preferably, 3).
5.4 Preservatives
It may be beneficial when storing ECL assay buffers to include a
preservative that prevents microbial growth. Preferably, the
preservative has little or no effect on ECL generated using the ECL
assay buffer, especially when using the ECL assay buffer on a
carbon based electrode. Azide has been found to be a suitable
preservative. Isothiazolones (e.g., Kathon,
2-methyl-4-isothiazolin-3-one and
5-chloro-2-methyl-4-isothiazolin-3-one), oxazolidines (e.g., Oxaban
A or 4,4 dimethyl oxazolidine) and related preservatives are
especially preferred due their compatibility with ECL, their high
activity and the low degree of problems associated with safety
hazards or environmental concerns.
5.5 Anti-Foam Agents
It may be beneficial, especially in HTS applications, to avoid the
production of bubbles or foam. For this reason it may be desirable
to add anti-foaming agents to ECL assay buffers. Applicants have
found that many commercial anti-foaming agents (including Antifoams
o-30, Antifoam 204, Antifoam A, Antifoam SE-15, Antifoam SO-25 and
Antifoam 289) may be added to ECL assay buffers without
significantly affecting the performance of the ECL assay
buffers.
5.6 ECL Labels
The compositions of the invention may include ECL labels. The ECL
labels may be conventional ECL labels. Examples of ECL labels
include: i) organometallic compounds where the metal is selected
from, for example, the noble metals of group VIII, including
Ru-containing and Os-containing organometallic compounds such as
the tris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and
related compounds. Preferably, the ECL labels are capable of
repeatedly emitting electrochemiluminescence. Preferred ECL labels
are ruthenium or osmium-containing organometallic species. More
preferably, these ruthenium or osmium-containing organometallic
comprise ruthenium or osmium chelated to polypyridyl ligands (most
preferably, bipyridine, phenanthroline, and/or substituted
derivatives thereof). Most preferably, the ECL labels comprise
ruthenium-tris-bipyridine, the bipyridine ligands being, optionally
substituted, e.g., with a linking group for attaching the label to
an assay reagent.
The ECL label may be linked to an assay reagent, optionally through
a linking group. Examples of binding reagents that may be linked to
an ECL label include: whole cells, cell surface antigens,
subcellular particles (e.g., organelles or membrane fragments),
viruses, prions, dust mites or fragments thereof, viroids,
antibodies, antigens, haptens, fatty acids, nucleic acids (and
synthetic analogs), proteins (and synthetic analogs), lipoproteins,
polysaccharides, inhibitors, cofactors, haptens, cell receptors,
receptor ligands, lipopolysaccharides, glycoproteins, peptides,
polypeptides, enzymes, enzyme substrates, enzyme products, second
messengers, cellular metabolites, hormones, pharmacological agents,
synthetic organic molecules, organometallic molecules,
tranquilizers, barbiturates, alkaloids, steroids, vitamins, amino
acids, sugars, lectins, recombinant or derived proteins, biotin,
avidin, streptavidin. The assay reagents are preferably useful as
binding reagents or enzyme substrates in, e.g., binding assays or
enzyme assays.
5.7 Compositions
One aspect of the invention relates to compositions comprising the
ECL assay buffers of the invention.
Another aspect of the present invention relates to compositions
suitable for use in an assay comprising a pH buffer substantially
free of inorganic phosphate. Suitable pH buffers include
glycylglycine ("Glygly"), tris(hydroxymethyl)aminomethane ("Tris")
or combinations thereof. Other pH buffers which are also
substantially free of or do not contain inorganic phosphate would
also be suitable for use in the invention.
According to one embodiment of the invention, the composition
comprises a pH buffer, wherein the composition is, preferably,
substantially free of inorganic phosphate and, preferably further
comprises one or more ECL co-reactants (preferably, TPA or
alternatively, a non-TPA coreactant, more preferably an
N-substituted morpholine or piperazine, most preferably PIPES).
According to a particularly preferred embodiment, the composition
is free of inorganic phosphate. Suitable pH buffers include glygly
and tris. Additional buffers may be selected on the basis of
certain preferred characteristics: i) the ability to buffer in the
pH range of 6.5-8.5, preferably 7-8 (more preferably, the pKa of
the buffer is in the range of 6.5 to 8.5 or more preferably, from
7.5 to 8.5); ii) commercial availability at low cost; iii) the lack
of an inhibitory effect on ECL and/or iv) the lack of a significant
oxidation wave in the range of 0-1.2 V or more preferably 0-1.5 V
(the voltage window for the oxidation of Ru(bpy).sub.3 and
TPA).
According to another embodiment of the invention, the composition
comprises a non-phosphate pH buffering agent and, preferably
further comprises one or more ECL co-reactants (preferably, TPA or
alternatively, a non-TPA coreactant, more preferably an
N-substituted morpholine or piperazine, most preferably PIPES).
Preferably, the composition has less than 15 mM inorganic
phosphate, more preferably it has less than 5 mM inorganic
phosphate, even more preferably it has less than 1 mM phosphate,
even more preferably it is substantially free of inorganic
phosphate, most preferably it is free of inorganic phosphate.
Suitable pH buffers include glygly and tris. Additional buffers may
be selected on the basis of certain preferred characteristics: i)
the ability to buffer in the pH range of 6.5-8.5, preferably 7-8
(more preferably, the pKa of the buffer is in the range of 6.5 to
8.5 or more preferably, from 7.5 to 8.5); ii) commercial
availability at low cost; iii)-the lack of an inhibitory effect on
ECL and/or iv) the lack of a significant oxidation wave in the
range of 0-1.2 V or more preferably 0-1.5 V (the voltage window for
the oxidation of Ru(bpy).sub.3 and TPA).
Preferably, the ECL co-reactant used in these embodiments is
suitable for use in an electrode induced luminescence reaction
(e.g., electrochemiluminescence). Suitable ECL co-reactants include
tripropylamine (TPA). Non-TPA coreactants (preferably, tertiary
amines other than TPA as described in the coreactants section
above) may be advantageous in some applications, in particular, in
non-washed assay formats.
Preferably, the composition comprises between 10 and 2000 mM pH
buffer, more preferably 50 and 1200 mM, even more preferably
between 100 and 600 mM, and most preferably between 300 and 500
mM.
Preferably, the composition comprises between 10 and 1000 mM, ECL
co-reactant, more preferably 30 and 600 mM, even more preferably
between 50 and 200 mM, and most preferably between 75 and 150
mM.
The optimal range of TPA concentrations in the pH buffers
containing Tris and Gly-Gly is very similar to concentrations of
ORIGENO .RTM.buffer (i.e., ranging from 50-200 mM). The tested
range of concentrations of Tris and Gly-Gly buffers is 100-600 mM.
Preferably, the concentration is 200 mM. ECL assay buffers
comprising non-TPA coreactants of the invention (preferably, PIPES)
may include similar ranges of coreactant concentrations, although
in many applications the preferred range is 10-100 mM, most
preferably 20-50 mM.
According to another preferred embodiment, the final formulation of
the Gly-Gly/TPA buffer is: 200 mM Gly-Gly, 100 mM TPA, 0.1% Triton
at pH=7.8.+-.0.05.
According to another preferred embodiment, the final formulation of
the Gly-Gly/TPA buffer is: 50-1000 mM Gly-Gly, 50-1000 mM TPA, at
pH=7.8.+-.1. Preferably, the formulation also comprises 0.2%-2%
Triton X-100 and/or 20-500 mM salt.
According to another preferred embodiment, the final formulation
for the Tris/TPA buffer is: 200 mM Tris, 100 mM TPA, 0.1% Triton at
pH=7.8.+-.0.05.
According to another preferred embodiment, the final formulation
for the Tris/TPA buffer is: 50-1000 mM Tris, 50-1000 mM TPA, at
pH=7.8.+-.0.05. Preferably, the formulation also comprises 0.2%-2%
Triton X-100. and/or 20-500 mM salt.
According to another preferred embodiment, the final formulation
for the PIPES/Phos buffer is: 40-1000 mM phosphate (preferably,
potassium phosphate), 10-200 mM PIPES, at pH=7.8.+-.0.05.
Preferably, the formulation also comprises 0.2%-2% Triton
X-100.
Using Tris and Gly-Gly assay buffers significantly improved the
stability of phosphopeptide-anti-phosphopeptide complexes in
ECL-based Tyrosine Kinase assays. However, some dissociation of the
complexes was observed in Tris buffer, although at much slower
rates than in ORIGEN assay buffer. In the case of the Gly-Gly
buffer, ECL signal slowly increased, because no stop reagent was
introduced into assay solution to quench the enzymatic
reaction.
According to one preferred embodiment, the composition further
comprises a stop reagent (i.e., a reagent added to stop a reaction
or reduce interference with a reaction). Chelating agents such as
ethylenediaminetetraacetic acid (EDTA) are common stop reagents in
Mg-dependent kinase assays. EDTA binds Mg.sup.2+ ions that are
require for successful activation of ATP. The addition of 5 mM EDTA
into Gly-Gly assay buffer, for example, helps to stop residual
tyrosine kinase enzymatic activity. Dissociation of
phosphopeptide-anti-phosphopeptide complexes in Gly-Gly/TPA buffer
with 5 mM EDTA does not exceed 1% per 1 hour in a non-washed assay
format. At concentrations higher than 10 mM, EDTA may have a
negative effect on absolute value of ECL signal, but does not
compromise stability of ECL signal upon incubation in assay buffer.
Depending on desired final read volume in 96-well plates (100 .mu.l
or 250 .mu.l) and the type of assay (washed or non-washed)
formulation of Gly-Gly/TPA solution may be different.
Preferably, the composition has a pH ranging from 6 to 9, more
preferably from 7 to 8, even more preferably from 7.5 to 8 and most
preferably about 7.8. According to one preferred embodiment, the pH
is adjusted by addition of an acid or base, preferably KOH, more
preferably 10% KOH.
One embodiment of the invention relates to an ECL assay buffer
comprising:
(a) glycylglycine (Gly-Gly), preferably from 0.1 to 0.7 M, more
preferably 0.3 and 0.5 M, and most preferred about 0.2 M; and
(b) tripropylamine (TPA), preferably from 0.01 M to 0.3 M, more
preferably 0.05 to 0.2, and most preferred about 0.1 M.
Preferably, the assay buffer further comprises EDTA (preferably 1
to 10 mM, more preferably 5 mM). Preferably, the assay buffer has a
pH ranging from 6 to 9, more preferably from 7 to 8, even more
preferably from 7.5 to 8 and most preferably about 7.8. According
to one preferred embodiment, the pH is adjusted by addition of an
acid or base, preferably KOH, more preferably 10% KOH.
Another preferred embodiment of the invention relates to an ECL
assay buffer comprising:
(a) tris[hydroxymethyl)aminomethane (Tris), preferably from 0.1 to
0.7 M, more preferably 0.3 to 0.5 M and most preferred about 0.2 M;
and
(b) tripropylamine (TPA), preferably from 0.01 M to 0.3 M, more
preferably from 0.05 to 0.2 M and most preferred about 0.1 M.
Preferably, the assay buffer further comprises
ethylenediaminetetraacetic acid (EDTA), preferably 1 to 10 mM, more
preferably 5 mM. Preferably, the composition has a pH ranging from
6 to 9, more preferably from 7 to 8, even more preferably from 7.5
to 8 and most preferably about 7.8. According to one preferred
embodiment, the pH is adjusted by addition of an acid or base,
preferably KOH, more preferably 10% KOH.
Another preferred embodiment of the invention relates to ECL assay
buffers comprising coreactants other than TPA, preferably
trialkylamines presenting hydrophilic functional groups (as
described in the coreactants section). Preferably the coreactant is
PPA or PIPES, most preferably PIPES. The concentration of
coreactant is, preferably, between 10 and 800 mM, most preferably
between 10 and 200 mM, most preferably between 20 and 50 mM.
Preferably, the ECL assay buffer also comprises a pH buffering
agent, preferably, phosphate, Tris or Gly-Gly. The concentration of
the pH buffering agent is preferably between 0 and 800 mM, more
preferably between 0 and 400 mM, even more preferably between 20
and 200 mM and most preferably between 75 and 150 mM. Preferably,
the composition has a pH ranging from 6 to 9, more preferably from
7 to 8, even more preferably from 7.2 to 7.8 and most preferably
about 7.5. Preferably, the ECL assay buffer also includes a
substance with a phenyl ether moiety and/or a detergent, preferably
a non-ionic detergent, even more preferably a phenyl ether
containing detergent, most preferably Triton X-100. Preferably the
concentration of detergent is greater than 0.02%, more preferably
greater than 0.05 %, most preferably between 0.05 and 0.5%.
According to one preferred embodiment, the reagents or compositions
of the invention further comprise one or more detergents and/or
surfactants (e.g., classes of non-ionic detergents/surfactants
known by the trade names of Nonidet, Brij, Triton, Tween, Thesit,
Lubrol, Genapol, Pluronic, Tetronic, F108, and Span). Especially
preferred detergents include: Tween 20, Triton X-100, NP-40 and
Thesit.
Another preferred embodiment of the invention relates to reagent
compositions comprising the assay buffers described above in
concentrated form. Preferably, the reagent compositions can be
diluted, preferably with an aqueous solution, to result in an assay
buffer having the optimal concentration of ingredients for use in
an assay, preferably an ECL assay.
Another embodiment relates a dry reagent precursor comprising one
of the above described assay buffers in dry form. Preferably, the
dry reagent precursor can be combined with a solution, preferably
with an aqueous solution, to result in an assay buffer solution
having the optimal concentration of ingredients for use in an
assay, preferably an ECL assay.
Another aspect of the invention relates to a reagent containing one
or more pH buffers substantially free of inorganic phosphate
suitable for use in providing a composition for conducting an
assay, preferably a luminescence assay, more preferably a
chemiluminescence assay or an electrode induced luminescence assay,
and most preferred an electrochemiluminescence assay.
Another aspect of the invention relates to a reagent containing one
or more ECL assay background reducing agents (preferably,
non-phosphate pH buffering agents) suitable for use in providing a
composition for conducting an assay, preferably a luminescence
assay, more preferably a chemiluminescence assay or an electrode
induced luminescence assay, and most preferred an
electrochemiluminescence assay. Preferably, the reagent has less
than 15 mM inorganic phosphate, more preferably it has less than 5
mM inorganic phosphate, even more preferably it has less than 1 mM
phosphate, even more preferably it is substantially free of
inorganic phosphate, and most preferably it is free of inorganic
phosphate.
According to one embodiment, the reagent comprises an ECL assay
buffer reducing agent (preferably, a non-phosphate pH buffering
agent) and/or is substantially free of inorganic phosphate, and the
reagent is suitable for use in providing a composition for
conducting an ECL assay wherein electromagnetic radiation is
emitted by an assay composition comprising members selected from
the group consisting of:
(i) a metal-containing ECL moiety capable of being converted to an
excited state from which electromagnetic radiation;
(ii) an ECL co-reactant (preferably an amine or an amine moiety,
most preferably a tertiary amine, most preferably TPA) which when
oxidized forms a strong reducing agent; and
(iii) an electrolyte capable of functioning as a medium in which
said ECL moiety and said ECL co-reactant can be oxidized.
Preferably, said reagent comprises said pH buffer, said ECL
co-reactant and one of the other two members of said group
(i)-(iii).
Another aspect of the invention relates to assay compositions
comprising one or more binding reagents, enzymes and/or substrates
and the pH buffer of the invention.
Another aspect of the invention relates to compositions, reagents,
kits and methods for carrying out protein kinase and phosphorylase
assays and/or for measuring phospho-peptides, phospho-proteins, and
phospho-amino acids. One embodiment of the invention relates to a
composition comprising a pH buffer and a phospho-peptide specific
antibody, where the composition is substantially free of inorganic
phosphate. Preferably, the composition is free of inorganic
phosphate. Preferably, the composition further comprises a
phosphopeptide, phosphoamino acid and/or phosphylated protein that
binds the phospho-peptide specific antibody.
Preferably, the pH buffer is selected from the group consisting of
glycylglycine, tris[hydroxymethyl)aminomethane or combinations
thereof.
Preferably, the composition further comprises one or more
components selected from the group consisting of kinases and kinase
substrate. According to another embodiment, the compositions
comprise or one or more components selected from the group
consisting of phosphatase and phosphatase substrate.
Preferably, the composition has a pH between 6 to 9, preferably
between 7 to 8, more preferably from 7.5 to 8, and most preferred
about 7.8.
According to one preferred embodiment, the composition further
comprises one or more ECL co-reactants. Preferably, the ECL
co-reactant comprises an-amine or an amine moiety. More preferably,
the ECL co-reactant comprises tripropylamine (TPA).
According to another preferred embodiment, the composition further
comprises a stop reagent. Preferably, the stop reagent comprises
ethylenediaminetetraacetic acid (EDTA).
According to another preferred embodiment, the composition further
comprises an acid or base, preferably KOH.
According to one preferred embodiment, the reagents or compositions
of the invention further comprises one or more detergents and/or
surfactants (e.g., classes of non-ionic detergents/surfactants
known by the trade names of Brij, Triton, Tween, Thesit, Lubrol,
Genapol, Pluronic, Tetronic, F108, and Span).
Preferably, the composition comprises an inhibitor and/or an
enzyme, more preferably an inhibitor to and/or an enzyme for a
phosphorylating or dephosphorylating reaction.
Another embodiment of the invention relates to a composition
comprising a pH buffer and an ECL co-reactant, said composition
being substantially free of inorganic phosphate. Preferably, the
composition is free of inorganic phosphate.
Preferably, the pH buffer is selected from the group consisting of
glycylglycine, tris[hydroxymethyl)aminomethane or combinations
thereof.
Preferably, the composition comprises one or more components
selected from the group consisting of kinases and kinase substrate
or one or more components selected from the group consisting of
phosphatase and phosphatase substrate.
Preferably, the composition has a pH between 6 to 9, preferably
between 7 to 8, more preferably from 7.5 to 8, and most preferred
about 7.8.
Preferably, the ECL co-reactant comprises an amine or an amine
moiety. More preferably, the ECL co-reactant comprises
tripropylamine (TPA).
According to another preferred embodiment, the composition further
comprises a stop reagent. Preferably, the stop reagent comprises
ethylenediaminetetraacetic acid (EDTA).
According to another preferred embodiment, the composition further
comprises an acid or base, preferably KOH.
Preferably, the composition comprises an inhibitor and/or an
enzyme, more preferably an inhibitor to and/or an enzyme for a
phosphorylating or dephosphorylating reaction.
According to one preferred embodiment, the pH buffer is
glycylglycine and said composition further comprises
ethylenediaminetetraacetic acid (EDTA) and TPA. Preferably, the
composition also further comprises KOH and/or an ECL moiety.
Another embodiment of the invention relates to a reagent comprising
a pH buffer, wherein said pH buffer is substantially free of
inorganic phosphate and said reagent is suitable for use in
providing a composition for conducting an ECL assay wherein
electromagnetic radiation is emitted by an assay composition
comprising members selected from the group consisting of:
(i) a metal-containing ECL moiety capable of being converted to an
excited state from which electromagnetic radiation is emitted;
(ii) an ECL co-reactant which when oxidized forms a strong reducing
agent; and
(iii) an electrolyte capable of functioning as a medium in which
said ECL moiety and said amine or amine moiety can be oxidized.
Preferably, the reagent further comprises said pH buffer, the ECL
co-reactant (preferably an amine or amine moiety) and one of the
other two members of said group (i)-(iii).
Another embodiment of the invention relates to a reagent comprising
a pH buffer, wherein said pH buffer is substantially free of
inorganic phosphate and said reagent is suitable for use in
providing a composition for conducting an ECL assay wherein
electromagnetic radiation is emitted by an assay composition
comprising members selected from the group consisting of:
(i) a metal-containing ECL moiety capable of being converted to an
excited state from which electromagnetic radiation is emitted;
(ii) an ECL co-reactant which when oxidized forms a strong reducing
agent, wherein said ECL co-reactants is an amine or an. amine
moiety (preferably TPA); and
(iii) an electrolyte capable of functioning as a medium in which
said ECL moiety and said amine or amine moiety can be oxidized.
Another embodiment of the invention relates to a composition
comprising:
(a) a phospho-specific antibody;
(b) a reagent selected from the group consisting of phosphorylating
enzyme, a substrate to a phosphorylating enzyme or combinations
thereof; and
(b) a pH buffer,
where the composition is substantially free of, preferably free of
inorganic phosphate.
Preferably, the composition also comprises an ECL co-reactant
(e.g., TPA).
5.8 Kits
One aspect of the invention relates to kits comprising, in one or
more containers, one or more components of the ECL assay buffers of
the invention. These components may be combined, optionally with
additional reagents, to form the ECL assay buffers of the
invention. The kits may also comprise additional assay related
components such as ECL labels, ECL labeled assay reagents, enzymes,
binding reagents, electrodes, assay plates, etc.
Another aspect of the invention relates to kits containing, in one
or more containers, one or more ECL assay buffers that contain a
trialkylamine coreactant of the invention other than TPA.
Preferably, the kit is contained in one or more glass or plastic
containers, appropriately labeled with information regarding the
buffer contents and instructions regarding proper storage and use
in assay. Some or all of the components of the ECL assay buffer may
be stored in a dry state. The kits may further comprise other assay
related components such as enzymes, binding reagents, electrodes,
assay plates, etc.
Another aspect of the invention relates to kits containing, in one
or more containers, one or more ECL assay buffers that are
substantially free of inorganic phosphate and/or comprise ECL assay
buffer reducing agents (preferably, non-phosphate pH buffering
agents). Preferably, the kit is contained in one or more glass or
plastic containers, appropriately labeled with information
regarding the buffer contents and instructions regarding proper
storage and use in assay. Some or all of the components of the ECL
assay buffer may be stored in a dry state. The kits may further
comprise other assay related components such as ECL labels, ECL
labeled assay reagents, enzymes, binding reagents, electrodes,
assay plates, etc.
No formal study on shelf-life stability of Gly-Gly/TPA buffer has
been performed. However, using 3-4 month old assay buffer did not
affect assay performance. Applicants believe that the same
precautions should be used for Gly-Gly stability, for example, as
for ORIGEN assay buffer. Preferably, concentrations of divalent
ions in the solution are kept below the .mu.M level.
Preferably, the kit is adapted or suitable for performing an ECL
assay wherein electromagnetic radiation emitted by a composition is
detected, which kit contains, in one or more containers, a pH
buffer and the kit is, preferably, substantially free of inorganic
phosphate and/or comprises an ECL assay buffer reducing agent
(preferably, a non-phosphate pH buffering agent). This kit also
comprises at least one other component selected from the group
consisting of: (i) a metal-containing ECL moiety capable of being
converted to an excited state from which electromagnetic radiation
is emitted; (ii) an ECL co-reactant (preferably an amine or an
amine moiety) which when oxidized forms a strong reducing agent;
and (iii) an electrolyte capable of functioning as a medium in
which said ECL moiety and said ECL co-reactant (e.g., amine or
amine moiety) can be oxidized, said kit comprising at least one
separate component in which one or more members of the group
consisting of said ECL moiety (i), ECL co-reactant (ii), and
electrolyte (iii) is included.
Another aspect of the invention relates to kits for use in
conducting assays, preferably luminescence assays, more preferably
electrode induced luminescence assays, and most preferably
electrochemiluminescence assays, comprising, in one or more
containers, one or more pH buffers substantially free of inorganic
phosphate and at least one assay component selected from the group
consisting of: (a) at least one luminescent label (preferably
electrochemiluminescent label); (b) at least one ECL co-reactant;
(c) one or more phospho-specific binding reagents; (d) one or more
electrodes and/or magnetic beads; (e) one or more blocking
reagents; (f) preservatives; (g) stabilizing agents; (h) enzymes;
(i) detergents; (j) desiccants and/or (k) hygroscopic agents.
Preferably, the kit comprises the assay module having one or more
assay electrodes, preferably an assay plate, more preferably
multi-well assay plates and the assay component(s) in one or more,
preferably two or more, more preferably three or more containers
according to U.S. application Ser. Nos. 10/185,274 and 10/185,363,
entitled "Assay Plates, Reader Systems and Methods for Luminescence
Test Measurements", each filed on Jun. 28, 2002, hereby
incorporated by reference.
According to one embodiment, the kit comprises one or more of the
assay components in one or more multi-well plate wells, preferably
in dry form.
According to one embodiment, the assay components are in separate
containers. According to another embodiment, the kit includes a
container comprising binding reagents and stabilizing agents.
According to another embodiment, the well reagents may include
binding reagents, stabilizing agents. Preferably, the kits do not
contain any liquids in the wells.
One preferred embodiment relates to a kit for use in conducting
electrode induced luminescence assays (preferably
electrochemiluminescence assays) comprising an assay plate,
preferably a multi-well assay plate, one or more pH buffers and at
least one assay component selected from the group consisting of at
least one luminescent label (preferably electrochemiluminescent
label) and at least one electrochemiluminescence coreactant,
wherein said pH buffers comprise an ECL assay buffer background
reducing agent (preferably, a non-phosphate pH buffering agent) or
are substantially free of phosphate and/or said ECL coreactant is
not TPA (and is preferably a functionalized tertiary alkylamine,
most preferably PIPES).
Another embodiment relates to a kit comprising a multi-well plate
and a pH buffer and at least one electrode induced luminescent
label (preferably electrochemiluminescent label) and/or at least
one bioreagent and/or at least one blocking reagent (e.g., BSA),
where the kit comprises an ECL assay buffer background reducing
agent (preferably, a non-phosphate buffering agent), is
substantially free of inorganic phosphate and/or comprises an ECL
coreactant other than TPA (preferably a functionalized tertiary
alkylamine, most preferably PIPES).
According to one preferred embodiment, the kit comprises at least
one material selected from group consisting of intact cell, cell
lysate, cell fragment, cell membrane, membrane ghost, organelle,
organelle fragment, organelle membrane, virion, virion fragment,
virion membrane, liposome, detergent solubilized protein, detergent
solubilized lipid or combinations thereof.
According to another embodiment, the kit comprises a biomaterial
selected from the group consisting of plasma membrane fragments,
endosomes, clathrin-coated vesicles, endoplamic reticulum
fragments, synaptic vesicles, golgi fragments, membrane subdomains,
mitochondria, peroxisomes, lysosomes, liposomes, viral particles,
viral-induced membrane enclosed particles shed from cells, and
intact, organismally-derived lipid membrane bodies.
According to one preferred embodiment, the kit comprises at least
one bioreagent, preferably immobilized on the plate surface
selected from: antibodies, fragments of antibodies, proteins,
enzymes, enzyme substrates, inhibitors, cofactors, antigens,
haptens, lipoproteins, liposaccharides, cells, sub-cellular
components, cell receptors, viruses, nucleic acids, antigens,
lipids, glycoproteins, carbohydrates, peptides, amino acids,
hormones, protein-binding ligands, pharmacological agents,
luminescent labels preferably ECL labels) or combinations thereof.
Preferably, at least one bioreagent is adapted or selected to
binding to one or more membranes resulting in an electrode having
such immobilized membranes.
Preferably, the biomaterial comprises a lipid/protein layer which
contains at least one active protein selected from the group
consisting of: single transmembrane receptors with intrinsic
tyrosine kinase activity; non-tyrosine kinase transmembrane
receptors (e.g., transferrin receptor); G-protein coupled receptors
(GPCR); GPCR effector proteins (e.g., adenylate cyclase);
phosphoinositides (e.g., phosphatidy inositol 4,5 bisphosphate
(PIP.sub.2)); phospholipid or sphingolipid composition,
identification, or function (i.e., changes in phosphotidylserine
presence during apoptosis); organelle-bound GTPases/guanine
nucleotide exchange factors (GEFs)/GTPase activating proteins
(GAPs); cytokine/chemokine receptors; cell adhesion molecules
(e.g., VCAM, integrins); cytoplasmic peripheral membrane protein
kinases (e.g., src); intracellular protein kinase adaptor/docking
proteins (e.g., insulin receptor substrate 1, GRB2); ion channels
(e.g., nicotinic acetylcholine receptor, CFTR, etc.); passive
transporters (e.g., glucose); active (ATP-driven) transporters;
ion-linked transporters (e.g., Na+/glucose);
glycosyltranferases/glycoprotein modifying enzymes; nuclear
membrane fragments; and soluble receptors.
Preferably, the kit includes immobilized reagents that comprise
proteins, nucleic acids, or combinations thereof.
According to one preferred embodiment, the plurality of wells
includes at least two different bioreagents. For example, a well
may include two or more assay domains, wherein two or more assay
domains have different bioreagent
Preferably, the kit comprises at least one electrochemiluminescence
coreactant and/or at least one electrode induced luminescence label
(preferably electrochemiluminescent label).
According to another embodiment, the kit is adapted for multiple
assays. Preferably, the kit further comprises an additional assay
reagent for use in an additional assay, the additional assay
selected from the group consisting of radioactive assays, enzyme
assays, chemical calorimetric assays, fluorescence assays,
chemiluminescence assays and combinations thereof.
According to another embodiment, the kit comprises two or more,
preferably four or more, more preferably eight or more, more
preferably 15 or more and most preferably 25 or more assay modules
or plates. According to a preferred embodiment, the kit is
contained in a resealable bag or container (e.g., zip-lock
opening).
Preferably, the bag or container is substantially impermeable to
water. According to one preferred embodiment, the bag is a foil,
preferably an aluminized foil.
The packaging may be translucent, transparent or opaque.
Preferably, the plates are packaged in aluminum lined plastic
containers or bags containing a dry or inert atmosphere (e.g., the
bags may be sealed under an atmosphere of nitrogen or argon or the
bags may contain a dessicant). According to another embodiment, the
containers are vacuum sealed.
Preferably, the container contains 1 plate. According to another
embodiment, the container contains ten plates. According to another
embodiment, the container includes between 10 and 100 plates.
Preferably, the assay modules or plates are sterile and/or
substantially free of dust and other contaminants.
Preferably, the assay modules are also substantially sterile.
According to one embodiment, the kit is manufactured (at least in
part) and/or packaged in a "clean room" environment. Preferably,
the kit is manufactured (at least in part) and/or packaged in a
Class 100,000 clean room having <100,000 particles (the clean
room particle count using a 0.5 micron particle count number) per
cubic foot (or 3.53 million particles per cubic meter).
Preferably, the contaminant particle counts (particles less than
0.5 microns) of the kit is less than 60 million per square meter,
more preferably 30 million per square meter, even more preferably
less than 20 million, even more preferably less than 15 million and
most preferably less than 10 million.
Preferably, the non-volatile residue in deionized water is less
than 0.50 g/meter.sup.2, more preferably less than 0.25
g/meter.sup.2, even more preferably less than 0.15 g/meter.sup.2
and most preferably less than 0.10 g/meter.sup.2.
Preferably the contaminant ion concentration is less than 50 ppm,
more preferably less than 20 ppm, even more preferably less than 10
ppm, even more preferably less than 5 ppm, and most preferably less
than 1 ppm.
5.9 Methods
Another aspect of the present invention relates to methods of using
the improved buffers, reagents and/or compositions of the
invention.
One embodiment of the invention relates to a method for conducting
an electrochemiluminescence assay wherein electrochemiluminescence
is induced in the presence of an ECL-assay buffer of the invention.
Preferably, the electrochemiluminescence is induced using a
carbon-based electrode.
Another embodiment of the invention relates to a method for
measuring the quantity of an ECL label wherein the label is induced
to emit electrochemiluminescence in the presence of an ECL assay
buffer of the invention and the electrochemiluminescence is
measured so as to measure the quantity of the ECL label. Preferably
the electrochemiluminescence is induced using a carbon-based
electrode. Most preferably, the label is bound to or held in
proximity to the electrode.
Another embodiment of the invention relates to a method for
measuring the quantity or activity of an analyte wherein the
analyte reacts with, forms a complex with, or competes in a
specific binding interaction with a labeled substance that
comprises an ECL label, wherein the label is induced to emit
electrochemiluminescence in the presence of an ECL assay buffer of
the invention and the electrochemiluminescence is measured so as to
measure the quantity or activity of the analyte. Preferably the
electrochemiluminescence is induced using a carbon-based electrode.
Most preferably, the presence or activity of the analyte results in
the label being bound to or released from an electrode (e.g., via
the formation of a specific binding complex or via a the cleavage
or formation of a chemical bond).
One embodiment of the invention relates to a method for conducting
an electrochemiluminescence assay wherein electrochemiluminescence
is induced in the presence of a composition comprising a pH buffer
and an ECL co-reactant, said composition being substantially free
of inorganic phosphate and/or comprising an ECL assay buffer
background reducing agent (preferably, a non-phosphate pH buffering
agent).
Another embodiment of the invention relates to a method for
conducting an electrochemiluminescence assay wherein
electrochemiluminescence is induced in the presence of a
composition comprising a pH buffer and an ECL co-reactant, wherein
the ECL coreactant is a functionalized trialkylamine, preferably
PIPES.
Another embodiment of the invention relates to a method of
generating emission of electromagnetic radiation comprising: (a)
forming a composition comprising: (i) a metal-containing ECL moiety
capable of being converted to an excited state from which
electromagnetic radiation is emitted; (ii) an ECL co-reactant
(preferably an amine or amine moiety) which, when oxidized, forms a
strong reducing agent; (iii) an electrolyte capable of functioning
as a medium in which said ECL moiety and said ECL co-reactant
(e.g., amine or amine moiety) can be oxidized; and (iv) a pH
buffers, wherein said composition is substantially free of
inorganic phosphate, comprises an ECL background reducing agent
(preferably, a non-phosphate pH buffering agent) and/or said ECL
co-reactant is a functionalized tertiary alkylamine; (b) exposing
the composition under suitable conditions to an amount of
electrochemical energy effective to induce the composition to emit
electromagnetic radiation; and (c) detecting emitted
electromagnetic radiation.
Another embodiment of the invention relates to a method of
effecting a specific-binding assay, either qualitatively or
quantitatively, in a sample or composition comprising a pH buffer
substantially free of inorganic phosphate and a phospo-specific
antibody. Preferably, the sample or composition further comprises
an ECL co-reactant.
Another embodiment of the invention relates to a method of
effecting a specific-binding assay, either qualitatively or
quantitatively, in a well having one or more assay domains with
binding reagents immobilized thereon using composition comprising a
pH buffer substantially free of inorganic phosphate. Preferably,
the composition further comprises a phospho-specific antibody.
Another embodiment of the invention relates to a method of
effecting a specific-binding non-washed assay, either qualitatively
or quantitatively, in a well having one or more assay domains with
binding reagents immobilized thereon using composition comprising a
ECL assay buffer that is substantially free of inorganic phosphate
and/or comprises an functionalized trialkylamine ECL
coreactant.
Another embodiment of the invention relates to a method of
performing an assay comprising forming a complex comprising a
kinase product and a phospho-specific antibody, wherein said
complex is not exposed to inorganic phosphate.
Another embodiment of the invention relates to a method of
performing an assay comprising:
(a) forming a complex comprising a kinase product and a
phospho-specific antibody, wherein said complex is not exposed to
inorganic phosphate;
(b) inducing a metal-containing ECL moiety to emit electromagnetic
radiation; and
(c) detecting emitted electromagnetic radiation.
Preferably, the complex further comprises said metal-containing ECL
moiety.
Another embodiment of the invention relates to a method of
generating emission of electromagnetic radiation, which comprises
the steps of:
(a) forming a composition comprising a pH buffer, said composition
being substantially free of inorganic phosphate, and (i) a
metal-containing ECL moiety capable of being converted to an
excited state from which electromagnetic radiation is emitted; (ii)
an amine or amine moiety which, when oxidized, forms a strong
reducing agent; and/or (iii) an electrolyte capable of functioning
as a medium in which said ECL moiety and said amine or amine moiety
can be oxidized;
(b) exposing the composition under suitable conditions to an amount
of electrochemical energy effective to induce the composition to
emit electromagnetic radiation; and
(c) detecting emitted electromagnetic radiation.
Another aspect of the invention relates to improved assays. The
invention is useful, for example, in enabling the detection and/or
quantitation of one or more analytes of interest. These reactions
include, for example, antigen-antibody interactions,
ligand-receptor interactions, DNA and RNA interactions, enzymatic
reactions, and other known reactions. In certain embodiments, the
invention relates to and methods for qualitatively and
quantitatively detecting the presence of analytes of interest in a
multi-component sample or multi-component system. (See, U.S.
application Ser. No. 60/318,293, (Entitled: "Methods and Apparatus
for Conducting Multiple Measurements on a Sample" by Glezer et al.,
filed on even date herewith, hereby incorporated by reference.
One preferred aspect of the invention include methods involving one
or more of the following: (a) a phospho-specific antibody; (b)
assay involving capture reagents immobilized on a solid surface
comprising an electrode or adjacent an electrode; and/or (c) assays
involving low detection levels (and/or requiring high signal to
background ratio).
The embodiments of the invention can be used to test a variety of
samples which may contain an analyte or activity of interest. Such
samples may be in solid, emulsion, suspension, liquid, or gas form.
They may be, but are not limited to, samples containing or derived
from, for example, cells (live or dead) and cell-derived products,
immortalized cells, cell fragments, cell fractions, cell lysates,
organelles, cell membranes, hybridoma, cell culture supernatants
(including supernatants from antibody producing organisms such as
hybridomas), waste or drinking water, food, beverages,
pharmaceutical compositions, blood, serum, plasma, hair, sweat,
urine, feces, tissue, biopsies, effluent, separated and/or
fractionated samples, separated and/or fractionated liquids,
organs, saliva, animal parts, animal byproducts, plants, plant
parts, plant byproducts, soil, minerals, mineral deposits, water,
water supply, water sources, filtered residue from fluids (gas and
liquid), swipes, absorbent materials, gels, cytoskeleton, protein
complexes, unfractionated samples, unfractionated cell lysates,
endocrine factors, paracrine factors, autocrine factors, cytokines,
hormones, cell signaling factors and or components, second
messenger signaling factors and/or components, cell nucleus/nuclei,
nuclear fractions, chemicals, chemical solutions, structural
biological components, skeletal (ligaments, tendons) components,
separated and/or fractionated skeletal components, hair, fur,
feathers, hair fractions and/or separations, skin, skin samples,
skin fractions, dermis, endodermis, eukaryotic cells, prokaryotic
cells, fungus, yeast, antibodies, antibody fragments, immunological
factors, immunological cells, drugs, therapeutic drugs, oils,
extracts, mucous, fur, oils, sewage, environmental samples, organic
solvents or air. The sample may further comprise, for example,
water, organic solvents (e.g., acetonitrile, dimethyl sulfoxide,
dimethyl formamide, n-methyl-pyrrolidone or alcohols) or mixtures
thereof.
Analytes that may be measured include, but are not limited to,
whole cells, cell surface antigens, subcellular particles (e.g.,
organelles or membrane fragments), viruses, prions, dust mites or
fragments thereof, viroids, antibodies, antigens, haptens, fatty
acids, nucleic acids (and synthetic analogs), proteins (and
synthetic analogs), lipoproteins, polysaccharides, inhibitors,
cofactors, haptens, cell receptors, receptor ligands,
lipopolysaccharides, glycoproteins, peptides, polypeptides,
enzymes, enzyme substrates, enzyme products, second messengers,
cellular metabolites, hormones, pharmacological agents, synthetic
organic molecules, organometallic molecules, tranquilizers,
barbiturates, alkaloids, steroids, vitamins, amino acids, sugars,
lectins, recombinant or derived proteins, biotin, avidin,
streptavidin, or inorganic molecules present in the sample.
Activities that may be measured include, but are not limited to,
the activities of phosphorylases, phosphatases, esterases,
trans-glutaminases, nucleic acid damaging activities, transferases,
oxidases, reductases, dehydrogenases, glycosidases, ribosomes,
protein processing enzymes (e.g., proteases, kinases, protein
phophatases, ubiquitin-protein ligases, etc.), nucleic acid
processing enzymes (e.g., polymerases, nucleases, integrases,
ligases, helicases, telomerases, etc.), cellular receptor
activation, second messenger system activation, etc.
Whole cells may be animal, plant, or bacteria, and may be viable or
dead. Examples include plant pathogens such as fungi and nematodes.
The term "subcellular particles" is meant to encompass, for
example, subcellular organelles, membrane particles as from
disrupted cells, fragments of cell walls, ribosomes, multi-enzyme
complexes, and other particles which can be derived from living
organisms. Nucleic acids include, for example, chromosomal DNA,
plasmid NA, viral DNA, and recombinant DNA derived from multiple
sources. Nucleic acids also include RNA's, for example messenger
RNA's, ribosomal RNA's and transfer RNA's. Polypeptides include,
for example, enzymes, transport proteins, receptor proteins, and
structural proteins such as viral coat proteins. Preferred
polypeptides are enzymes and antibodies. Particularly preferred
polypeptides are monoclonal antibodies. Hormones include, for
example, insulin and T4 thyroid hormone. Pharmacological agents
include, for example, cardiac glycosides. It is of course within
the scope of this invention to include synthetic substances which
chemically resemble biological materials, such as synthetic
polypeptides, synthetic nucleic acids, and synthetic membranes,
vesicles and liposomes. The foregoing is not intended to be a
comprehensive list of the biological substances suitable for use in
this invention, but is meant only to illustrate the wide scope of
the invention.
The composition or reagent of the invention are preferably aqueous.
The composition or reagent can also be non-aqueous. Examples of
suitable organic liquids are acetonitrile, dimethylsulfoxide
(DMSO), dimethylformamide (DMF), methanol, ethanol, and mixtures of
two or more of the foregoing. Illustratively, tetraalkylammonium
salts, such as tetrabutylammonium tetrafluoroborate, are soluble in
organic liquids and can be used with them to form non-aqueous
electrolytes.
Also, typically, the analyte of interest is present at a
concentration of 10.sup.-3 molar or less, for example, at least as
low as 10.sup.-18 molar. The sample which may contain the analyte
of interest, can be in solid, emulsion, suspension, liquid, or gas
form, and can be derived from, for example, cells and cell-derived
products, water, food, blood, serum, hair, sweat, urine, feces,
tissue, saliva, oils, organic solvents or air. The sample can
further comprise, for example, water, acetonitrile, dimethyl
sulfoxide, dimethyl formamide, n-methyl-pyrrolidone or
alcohols.
In one embodiment, the reagent includes an ECL moiety conjugated to
an antibody, antigen, nucleic acid, hapten, small nucleotide
sequence, oligomer, ligand, enzyme, or biotin, avidin,
streptavidin, Protein A, Protein G, or complexes thereof, or other
secondary binding partner capable of binding to a primary binding
partner through protein interactions.
One embodiment of the invention relates to a method of detecting or
quantitating an analyte of interest by ECL assay, which
comprises:
(1) forming a composition comprising (a) a sample to be tested for
the analyte of interest, (b) at least one substance selected from
the group consisting of (i) additional analyte of interest or an
analog of the analyte of interest, (ii) a binding partner of the
analyte of interest or its said analog, and (iii) a reactive
component capable of binding with (i) or (ii), (c) a
metal-containing ECL moiety capable of being converted to an
excited state from which electromagnetic radiation is emitted, said
ECL moiety being capable of entering into a binding interaction
with the analyte of interest or a substance defined in (b)(i),
(b)(ii), or (b)(iii); (d) an ECL co-reactants (preferably an amine
or an amine moiety) which, when oxidized, forms a strong reducing
agent, and (e) an electrolyte capable of functioning as a medium in
which said ECL moiety and said species can be oxidized;
(2) exposing said composition to an amount of electrochemical
energy effective to induce the composition-to emit electromagnetic
radiation; and
(3) detecting emitted electromagnetic radiation, wherein the sample
is not exposed to inorganic phosphate detrimental to the
performance of the assay or wherein said composition further
comprises an ECL assay buffer background reducing agent
(preferably, a non-phosphate pH buffering agent). Preferably, the
composition has less than 15 mM inorganic phosphate, more
preferably it has less than 5 mM inorganic phosphate, even more
preferably it has less than 1 mM phosphate, even more preferably it
is substantially free of inorganic phosphate, most preferably it is
free of inorganic phosphate.
Solid phase assay formats (e.g., solid phase binding assays) often
couple a biological activity or binding reaction to attachment or
dissociation of a label from a surface. For example the binding
interaction between a binding reagent that is attached and a
labeled analyte results in the localization of the label on the
solid phase supporting the immobilized binding reagent. The
biological activity or binding reaction to be measured can be
quantified through a measurement of the labels on the solid phase.
Many solid phase assay formats involve a wash step to remove
unbound labels prior to detecting labels on the solid phase (washed
assays). Assays without this wash step can be achieved when the
detection method can discriminate between free and bound labels.
Non-wash assay formats are desired because washing steps, in many
applications, can be difficult or cumbersome to carry out. In many
cases, however, the performance of non-wash assays is limited by
high background signals due to incomplete discrimination of free
vs. bound labels.
We have found, surprisingly, that the ECL assay buffers of the
invention improve the discrimination of free vs. bound labels in
ECL assays using assay reagents attached (e.g., by covalent
interactions, specific binding interaction, non-specific
adsorption, etc,) to the working electrode used to induce ECL (i.e,
the ability to selectively detect labels that are bound to the
electrode). More specifically, the compositions and reagents of the
invention improve the ratio of ECL signal from bound label to ECL
signal from free label. It is believed that the ECL assay buffers
of the invention decrease the distance from the solid electrode
surface from which an ECL label is induced to emit luminescence.
This, in turn, increases the signal of bound label (which may be
bound to the electrode surface) vs. free label (which is not bound
to the electrode). Another way to characterize this is in terms of
an "effective excitation length"--the maximum distance at which a
free ECL label is able to be excited. The "effective excitation
length" is impacted by i) the distance short-lived intermediates
involved in the generation of ECL (e.g., oxidation product of TPA)
can diffuse from the electrode before they are destroyed in a
destructive side reaction (a function of the lifetimes and
diffusion constants for these intermediates) and ii) the rate at
which free labels or labeled reagents diffuse into the region close
enough to the electrode to participate in a reaction with these
reactive intermediates (a function of the diffusion constant for
the unbound ECL labels or labeled reagents).
Using the ECL assay buffers of the invention, the effective
excitation length is reduced by >50%, preferably by >75%,
even more preferably by >90%. Thus, the ECL assay buffers of the
invention are desirable since they maximize the ratio of bound/free
ECL signal which enhances the performance of the assay. These
considerations are particularly important for measuring low
affinity interactions, which require the presence of the labeled
species in high concentration in the solution but would also be
expected to suffer from significant signal loss due to binding
complex dissociation during wash steps.
Accordingly, another aspect of the invention relates to non-wash
format assays using pH buffer substantially free of inorganic
phosphate which maximizes the ratio of bound/free ECL signal.
Preferably, the assay involves the capture of an ECL label at a
surface having or being adjacent to an electrode surface. See, for
example, U.S. Pat. Nos. 6,066,448; 6,090,545; 6,140,045; 6,207,369,
6,214,369; and U.S. application Ser. Nos. 10/185,274 and
10/185,363, entitled "Assay Plates, Reader Systems and Methods for
Luminescence Test Measurements", each filed on Jun. 28, 2002,
hereby incorporated by reference.
Thus, another embodiment of the invention relates generally to
electrochemiluminescence assays using reagents immobilized on a
surface (preferably an electrode surface) and having advantageous
effective excitation lengths. Preferably, the assay results in an
effective excitation length less then 100 microns, more preferably
less than 75 microns, even more preferably less than 50 microns,
even more preferably less than 25 microns, even more preferably
less than 10 microns, even more preferably less than 5 microns and
most preferably less than 1 micron. According to a particularly
preferred, embodiment, the effective excitation length is less than
0.5 micron, preferably less than 0.2 microns, even more preferably
less than 0.1 micron.
5.10 Systems
Yet another aspect of the present invention relates to system for
performing assays and comprising or using the reagents and/or
compositions of the invention.
One embodiment of the invention relates to a system for ECL
detection or quantitation of an analyte of interest in a sample,
said system comprising:
(a) a pH buffering agent;
(b) a sample;
(c) at least one substance selected from the group consisting of:
(i) added analyte of interest or an analog of the analyte of
interest, (ii) a binding partner of the analyte of interest or its
said analog, and (iii) a reactive component capable of binding with
(i) or (ii),
wherein one of said substances is linked, either directly or
through one or more other molecules, to a metal-containing ECL
moiety which is capable of being converted to an excited state from
which electromagnetic radiation is emitted;
(d) an ECL co-reactant, preferably an amine or amine moiety, which
is capable of being converted to a strong reducing agent and an
electrolyte;
(d) one or more electrodes for inducing the ECL moiety to emit
electromagnetic radiation; and
(e) one or more detectors for measuring the emitted radiation to
determine the presence or quantity of the analyte of interest in
the sample;
Wherein said pH buffering agent is substantially free of phosphate
or is an ECL assay buffer background reducing agent and/or said ECL
coreactant is a functionalized tertiary amine.
Another embodiment of the invention relates to a system for ECL
detection or quantitation of an analyte of interest in a sample,
said system comprising,
(a) a pH buffering agent;
(b) a sample;
(c) at least one substance selected from the group consisting of:
(i) added analyte of interest or an analog of the analyte of
interest, (ii) a binding partner of the analyte of interest or its
said analog, and (iii) a reactive component capable of binding with
(i) or (ii),
wherein one of said substances is linked, either directly or
through one or more other molecules, to a metal-containing ECL
moiety which is capable of being converted to an excited state from
which electromagnetic radiation is emitted;
(d) an ECL co-reactant, preferably a functionalized tertiary amine,
which is capable of being converted to a strong reducing agent and
an electrolyte;
(d) one or more electrodes for inducing the ECL moiety to emit
electromagnetic radiation; and
(e) one or more detectors for measuring the emitted radiation to
determine the presence or quantity of the analyte of interest in
the sample.
5.11 Method of Selecting Biologically Active Compounds and
Producing Novel Drugs
Another aspect of the invention relates to improved methods and
systems for selecting or identifying biologically active compounds
and, optionally, incorporating such biologically active compounds
into suitable carrier compositions in appropriate dosages as
described in paragraph 6.11 of U.S. application Ser. Nos.
10/185,274 and 10/185,363, entitled "Assay Plates, Reader Systems
and Methods for Luminescence Test Measurements", each filed on Jun.
28, 2002, hereby incorporated by reference.
One embodiment relates to the use of the invention to screen for
new drugs, preferably, by high-throughput screening (HTS),
preferably involving screening of greater than 50, more preferably
100, more preferably 500, even more preferably 1,000, and most
preferably 5,000. According to a particularly preferred embodiment,
the screening involves greater than 10,000, greater than 50,000,
greater than 100,00, greater than 500,000 and/or greater than
1,000,000 compounds.
Advantageously, the reagents, compositions, methods, apparatus
and/or assay plates or modules of the invention may be integrated
into and/or used in a variety of screening and/or drug discovery
methods. Such screening and/or drug discovery methods include those
set forth in U.S. Pat. No. 5,565,325 to Blake; U.S. Pat. No.
5,593,135 to Chen et al.; U.S. Pat. No. 5,521,135 to Thastrup et
al.; U.S. Pat. No. 5,684,711 to Agrafiotis et al.; U.S. Pat. No.
5,639,603 to Dower et al.; U.S. Pat. No. 5,569,588 to Ashby et al.;
U.S. Pat. No. 5,541,061; U.S. Pat. No. 5,574,656; and U.S. Pat. No.
5,783,431 to Peterson et al.
According to another embodiment, the invention further comprises
identifying adverse effects associated with the drug and storing
information relating to the adverse effects in a database. See,
U.S. Pat. No. 6,219,674 by Classen.
Another aspect of the invention relates to improved biologically
active compounds and/or drugs made using the inventive methods.
6. EXAMPLES
The following examples are illustrative of some of the electrodes,
plates, kits and methods falling within the scope of the present
invention. They are, of course, not to be considered in any way
limitative of the invention. Numerous changes and modification can
be made with respect to the invention by one of ordinary skill in
the art without undue experimentation.
Example I
ECL Measurements
Unless otherwise indicated, ECL measurements were carried out using
multi-well plates having integrated carbon ink electrodes (see,
Example 6.1 and, in particular, Plate Types A and B of copending
U.S. application Ser. Nos. 10/185,274 and 10/185,363, each filed on
Jun. 28, 2002, entitled "Assay Plates, Reader Systems and Methods
for Luminescence Test Measurements", hereby incorporated by
reference). The electrodes were, optionally treated with an oxygen
plasma prior to being coated with binding reagents (plasma treated
and non-plasma treated plates, respectively, are designated
hereafter as PT or NPT plates). Binding reagents were immobilized
on the working electrodes of the plates using the methods described
in the U.S. application Ser. Nos. 10/185,274 and 10/185,363 or
adaptations thereof. Unless otherwise indicated, ECL measurements
were carried out using multi-well plate readers adapted for use
with these multi-well plates. The readers and their use are
described in Example 6.3 of the U.S. application Ser. Nos.
10/185,274 and 10/185,363. U.S. application Ser. Nos. 10/185,274
and 10/185,363 and, in particular, the descriptions of plate types,
immobilization methods, plate readers and ECL measurement methods,
are hereby incorporated by reference. The reported ECL intensities
are reported in relative terms and may depend on the instrument,
gain settings and plates used in a particular experiment. For this
reason, the absolute values reported in different experiments may
not be directly comparable.
Example II
Tyrosine Kinase Assay
The format involved the phosphorylation of a kinase substrate
immobilized on electrodes in multi-well plates adapted for ECL
measurements (see Example I), complexation of the product to a
labeled anti-phosphotyrosine antibody and detection of the
surface-bound label via an ECL measurement in the presence of an
ECL Assay Buffer comprising an ECL coreactant. The electrodes were
pretreated by etching in an oxygen plasma to increase the amount of
exposed carbon: The kinase substrate--poly(Glu, Tyr) having a 4:1
ratio of Glu to Tyr and a molecular weight of 20,000-50,000 Daltons
(PGT, Sigma-Aldrich Co.)--was immobilized by non-specific
adsorption on the surface of the working electrodes in the wells of
the plates. The working electrode in each well was treated with
1500 nL of a solution containing 1 mg/ml PGT in PBS buffer. The
plate was then dried overnight and blocked in a 5% solution of
bovine serum albumin at 4.degree. C. The plate was washed to remove
the blocking agent prior to use.
The assay was carried out by adding, to each well, 50 .mu.L of a
buffered solution containing a soluble tyrosine kinase (c-src,
Upstate Biotechnology), an anti-phosphotyrosine monoclonal antibody
(Abzyme, IGEN International) that was labeled with a sulfonated
derivative of ruthenium-tris-bipyridine (Sulfo-TAG.TM. label, Meso
Scale Discovery), ATP and Mg.sup.+2. The reaction was allowed to
proceed for 1 hour. The plate was washed and 100 .mu.L of an ECL
Assay Buffer containing tripropylamine was added. The plate was
analyzed using electrochemiluminescence detection as described in
Example I.
When a conventional ECL Assay Buffer containing TPA in a phosphate
buffer (ORIGEN Assay Buffer, IGEN International) was used in the
protocol, the complex formed between the labeled antibody and the
phosphorylated substrate dissociated over a period of 30 min. to an
hour (the majority of the dissociation occurring within the first
few minutes) due to the competitive binding of phosphate ions with
the labeled antibodies. One approach to avoiding this problem is to
control the time of exposure of the formed complex to the inorganic
phosphate-containing solution. This approach, however, may be
impractical in some assays such as high throughput assays involving
large numbers of plates.
Applicants discovered another solution to overcome the problem was
the use of phosphate-free buffers. Surprisingly, it was discovered
that the phosphate could be replaced with other buffers without
compromising the ability of the ECL Assay Buffers to support the
generation of ECL.
Assays were carried out using the following two ECL Assay Buffer
compositions:
Gly-Gly Assay Buffer:
0.4 M Glycylglycine (Gly-Gly) 0.1 M Tripropylamine (TPA) 12 mM
Ethylenediaminetetraacetic Acid (EDTA) pH=7.8 (adjusted by addition
of 10% KOH) Tris Assay Buffer: 0.4 M
Tris(hydroxymethyl)aminomethane (Tris) 0.15 M Tripropylamine (TPA)
12 mM Ethylenediaminetetraacetic Acid (EDTA)
pH=7.8 (adjusted by addition of 10% KOH)
EDTA was added into the new ECL Assay Buffers to stop the
phosphorylation reaction by sequestering Mg.sup.+2, an ion required
for kinase activity (EDTA was not required in phosphate-based ECL
Assay Buffers due to the affinity of phosphate for magnesium ions).
This composition allowed us to combine two steps (addition of
stop-solution and actual assay buffer) into one step. Applicants
found that EDTA interfered with ECL generation at concentrations
higher that 10 mM, but that 5-12 mM EDTA was enough to stop the
reaction while only causing a small decrease in ECL signal.
FIG. 1 shows the decrease in ECL from the phosphopeptide-antibody
complex as a function of the time between the addition of the Assay
Buffer and the measurement of ECL. Surprisingly, while exposure of
the complex to the phosphate-containing ORIGEN Assay Buffer led to
almost complete dissociation of the complex (within the time it
took to put the plate into the ECL reader for the 0 min. point),
the complex showed excellent stability in the Gly-Gly (<80 %
dissociation over 40 min) and Tris (<55% dissociation over 40
min) Assay Buffers.
The stability of the complex was improved further by eliminating
the wash step prior to addition of the assay buffer. FIG. 2 shows
the results of an experiment in which 200 .mu.L of Gly-Gly Assay
Buffer (as described above except that the concentration of EDTA
was 5 mM and 0.2% Triton X-100 was added) was added directly to the
reaction mixture without an intervening wash step. Storage of the
plates for 20 hours prior to measuring ECL resulted in only a 15%
decrease in signal.
One additional surprising advantage of the protocol was its
robustness. Surprisingly, the time of the phosphorylation step was
the only time that required tight control in order to get
reproducible results. The results of the assay did not depend on
the time between all other steps.
Example III
Detection of Phosphorylated EGF Receptor
A sandwich immunoassay was used to detect autophosphorylated
.alpha.-epidermal growth factor receptor (.alpha.-EGFR) in cell
lysates prepared from EGF-activated A-431 cells (American Type
Culture Collection). The assay employed a biotin labeled capture
antibody directed against the .alpha.-EGFR extracellular domain and
a Sulfo-TAG labeled detection antibody that is specific for
phosphotyrosine (see Example II). The biotin-labeled antibody was
immobilized on the working electrode of multi-well plates adapted
for use in ECL assays (see Example I) through the interaction of
the biotin label with avidin that was passively adsorbed on the
electrode surface. Solubilized EGFR (in RIPA, a
deoxycholate-containing buffer) was then added and allowed to bind
to the anti-EGFR antibody. Subsequently, the Sulfo-TAG labeled
.alpha.-phosphotyrosine antibody was added to detect
autophosphorylated EGFR.
In an end-product stability experiment, an assay was carried out as
described above and, prior to the induction and measurement of ECL,
the resulting sandwich complex was incubated for varying amounts of
time in two different ECL Assay Buffers: 150 mM TPA/150 mM
Phosphate, pH 7.48 and 100 mM TPA/400 mM glycine-glycine, pH 7.8.
FIG. 3 shows that there was a significant time-dependent decay in
signal in the presence of the phosphate-containing buffer; the
signal decreased by roughly 80% after one hour. The glycine-glycine
buffer reduced this decrease to roughly 20%. The great loss of
signal that occurs in the phosphate buffer is believed to be due to
the phosphate ion competing with the phosphorylated protein for the
anti-phosphotyrosine antibody. Moreover, the signal to background
ratio was increased 2.5 fold using the glycyl-glycine assay
buffer.
Example IV
Effect of ECL Assay Buffer Composition on the Ability to
Discriminate between Specific Signal and Assay Buffer
Background
In many ECL assay formats, the sensitivity with which an ECL label
can be measured is limited by the light signal (and the noise in
the light signal) generated by the ECL Assay Buffer in the absence
of the ECL label (ECL Assay Buffer background). This limitation is
especially evident in washed assays, assays exhibiting low levels
of non-specific binding and/or assays employing ECL readers having
sensitive, low noise, light detectors. Applicants examined the
relationship between ECL Assay Buffer composition and the ability
to discriminate between the signal due to an ECL label and the ECL
Assay Buffer background. In particular, applicants tested four ECL
Assay Buffer formulations that varied in the identity of the ECL
coreactant and/or the identity of the pH buffering agent:
TPA/Phosphate, TPA/Tris, TPA/Gly-Gly and PIPES/Phosphate (where
PIPES stands for 1,4-piperazine-1,4-bis(2-ethanesulfonic acid).
The experiments were carried out on multi-well plates (as described
in Example I) that had avidin immobilized on the working
electrodes. The experiments were carried out on plates that were
treated with an oxygen plasma (PT plates) as well as on untreated
plates (NPT plates). Avidin was immobilized on PT plates by
dispensing 2.5 .mu.L of solution containing 0.5 mg/mL avidin and
0.0035% Triton X-100 on the working electrode of each well,
allowing the solution to evaporate to dryness over a period of 1
hour and blocking the remaining surfaces of the well overnight at
4.degree. C. with a 5% (w/v) solution of BSA. Avidin was
immobilized on NPT plates by dispensing 2.5 .mu.L of solution
containing 0.5 mg/mL avidin and 0.0075% Triton X-100 on the working
electrode of each well, allowing the solution to evaporate to
dryness overnight and blocking the remaining surfaces of the well
for 2 hours with a 5% (w/v) solution of BSA. Varying amounts of an.
ECL label could be brought into proximity with the electrode
surface by treating the wells with a solution containing bovine IgG
that was labeled with biotin and .about.1.9 Sulfo-TAG labels per
protein (BT-IgG*). The binding of the BT-IgG* was accomplished by
adding 50 .mu.L of a solution containing 1 nM of BT-IgG* in PBS to
the wells and incubating for a period of 60 minutes while shaking.
The wells were washed with water, 100 .mu.L of ECL Assay Buffer was
added and ECL was measured. The signal due to ECL Assay Buffer
Background was measured by repeating the experiment as described
above except that the BT-IgG* was omitted.
The four combinations of coreactant and pH buffer to be tested were
optimized to identify the concentration of coreactant, the
concentration of pH buffer and the pH that gave the best ratio of
signal from BT-IgG* to ECL Assay Buffer background (S/B ratio). The
concentrations of coreactant were varied from 25 to 200 mM for TPA
or 13 to 200 mM for PIPES. The concentrations of pH buffer were
varied from 50 to 300 mM for phosphate, 100-600 mM for Tris or
50-800 mM for Gly-Gly. The pH was varied from 7 to 8. In all cases,
the ECL Assay Buffers also contained 0.05% Triton X-100. KOH or HCl
were added as necessary to achieve the desired pH. Within the
ranges tested, all the formulations gave adequate performance for
use in ECL assays, however, the following optimized formulations
were identified on the basis of their S/B ratios: TPA/Phosphate
(125 mM TPA, 200 mM phosphate, 0.05% Triton X-100, pH 7.5);
TPA/Tris (125 mM TPA, 200 mM Tris, 0.05% Triton X-100, pH 7.8);
TPA/Gly-Gly (100 mM TPA, 200 mM Gly-Gly, 0.05% Triton X-100, pH
7.8) and PIPES/Phos (25 mM PIPES, 100 mM phosphate, 0.05% Triton
X-100, pH 7.5).
FIGS. 4A and 4B compare the performance of the four optimized
formulations for assays carried out on NPT plates and PT plates,
respectively. We found that the TPA/Phosphate and TPA/Tris buffers
gave roughly comparable signals for the BT-IgG*, however, the lower
ECL Assay Buffer Background of the TPA/Tris system led to a
significant improvement in the S/B ratio relative to the
TPA/Phosphate buffer. Assuming the noise in the background to be
roughly proportional to the background signal, the 4-6 fold
improvement in S/B ratio transfers directly to a 4-6 fold
improvement in detection limits. Despite having lower specific
signals, the TPA/Gly-Gly buffer had an S/B ratio that was
approximately the same as the TPA/Tris buffer and could, therefore,
be expected to produce similar detection limits. The
PIPES/Phosphate buffer performed slightly better (in terms of S/B
ratio) than the TPA/Phosphate buffer on unetched plates and
slightly worse on etched plates.
Example V
Effect of the ECL Assay Buffer Composition on the Ability to
Discriminate between ECL Labels that are Bound to an Electrode
Surface and ECL Labels that are Free in Solution
In some ECL assay formats, the sensitivity with which an ECL label
held in proximity to an electrode can be measured is limited by the
light signal (and the noise in the light signal) generated by ECL
labels in solution. This limitation is especially evident in assays
in which labels in solution are not removed by washing prior to the
addition of an ECL Assay Buffer and the measurement of ECL
(Unwashed Assays). Applicants examined the relationship between ECL
Assay Buffer composition and the ability to discriminate between
the signal due to ECL labels bound to (or held in proximity to) an
electrode and ECL labels that are free in solution.
In these experiments, the specific signal from bound ECL labels was
measured using BT-IgG* bound to avidin-coated electrodes as
described in Example IV. The ECL. Assay Buffer background was
determined by omitting the BT-IgG*, also as described in Example
IV. The ECL signal from free ECL labels in solution was determined
similarly to the ECL Assay Buffer background except that the ECL
Assay Buffer added to the wells included 10 nM bovine IgG having
3.9 labels per protein (IgG*). The ratio of the ECL signal from the
bound BT-IgG* to the ECL signal from the free IgG* (B/F ratio) is
indicative of the sensitivity with which bound ECL labels can be
detected in the presence of free ECL labels in solution.
The four optimized ECL Assay Buffers from Example IV were tested
for their ability to discriminate between surface bound ECL labels.
The results are presented in Tables IA and IB. The replacement of
phosphate with Tris led to some improvement in the B/F ratio for
TPA-containing buffers. The most drastic improvement, however, was
achieved by substituting the coreactant component, i.e., by
replacing TPA with PIPES. There was a 4-5 fold improvement in the
B/F ratio by replacing TPA/Phos with PIPES/Phos.
TABLE-US-00001 TABLE IA ECL signal measured on avidin-coated PT
plates from bound BT-IgG* (bound from a 1.5 nM solution), free IgG*
(present in a 1.5 nM solution) and ECL Assay Buffer Background. S/B
= (Bound)/(Background); B/F = (Bound-Background)/(Free-Background).
Free Bound BT-IgG* IgG* Background S/B B/F TPA/Phosphate 77493 2267
305 254 39 TPA/Tris 83167 1873 61 1363 46 TPA/Gly-Gly 28168 1111 35
805 26 PIPES/Phosphate 64724 670 319 203 183
TABLE-US-00002 TABLE IB ECL signal measured on avidin-coated NPT
plates from bound BT-IgG* (bound from a 1.5 nM solution), free IgG*
(present in a 1.5 nM solution) and ECL Assay Buffer Background. S/B
= (Bound)/(Background); B/F = (Bound-Background)/(Free-Background).
Free Bound BT-IgG* IgG* Background S/B B/F TPA/Phosphate 264,063
4671 464 569 63 TPA/Tris 270,809 2734 89 3043 102 TPA/Gly-Gly
123,393 1663 38 3247 76 PIPES/Phosphate 172,226 728 164 1050
305
PIPES-containing ECL Assay Buffers were prepared with phosphate,
Tris or Gly-Gly as the buffering agent. The B/F ratio of each of
these mixtures was further optimized by varying the concentration
of PIPES and the buffer component. The concentration of PIPES was
varied from 12.5 to 200 mM in the phosphate-based buffer and 25 to
100 mM in the Tris and Gly-Gly buffers. The concentrations of the
buffering agent were varied from 100 to 400 mM. In all cases, the
ECL Assay Buffers also contained 0.05% Triton X-100. KOH or HCl
were added as necessary to achieve the desired pH. Within the
ranges tested, all the formulations gave adequate performance for
use in ECL assays including compositions that had no added buffer
component. It was also possible to omit the buffering agent and
achieve adequate performance due the ability of PIPES to act-as a
pH buffer. PIPES concentrations of 20-100 mM were found to provide
high B/F ratios while maintaining reasonable ECL intensities. The
best performance was achieved when the phosphate concentrations was
roughly 2-4 times the PIPES concentration. The following optimized
formulations were identified on the basis of having both high S/B
ratios and reasonable signal intensities: PIPES/PHOSPHATE (25 mM
PIPES, 100 mM phosphate, 0.05% Triton X-100, pH 7.5); PIPES/Tris
(25 mM PIPES, 200 mM Tris, 0.05% Triton X-100, pH 7.8) and
PIPES/Gly-Gly (25 mM PIPES, 100 mM Gly-Gly, 0.05% Triton X-100, pH
7.8).
FIGS. 5A and 5B compare the performance of the three optimized
formulations for nonwashed assays carried out on NPT plates and PT
plates, respectively. The figures compare the performance to the
conventional TPA/Phosphate buffer. We found that for all three
buffering agents that were tested, the use of PIPES as a coreactant
led to significant improvements (as much as factors of 4-5) in the
B/F ratio relative to TPA/Phosphate.
Example VI
Effect of Detergent on the Performance of ECL Assay Buffers
FIG. 6 shows the effect of the presence of various non-ionic
detergents on ECL signal from BT-IgG* bound to avidin-coated plasma
treated electrodes. The detergents were added at 0.5 %(w/v) to one
of two ECL Assay Buffers: FIG. 6A shows the results obtained with
150 mM TPA, 250 mM phosphate, pH 7.5; FIG. 6B shows the results
obtained with 50 mM PIPES, 150 mM phosphate, pH 7.5. BT-IgG* (50
.mu.L of a 0.01 mM solution) was allowed to bind to the avidin
surface. The plates were washed, ECL Assay Buffers were added and
the plates were analyzed using ECL detection. The figure shows the
Assay Buffer background, signal and S/B ratio (calculated as in
Example 4) measured using each of the ECL Assay Buffers. Of the
detergents tested, only Triton X-100 had a significant effect on
performance. For the PIPES-based buffer, the effect was large;
addition of Triton led to a >2.5 fold increase in the S/B ratio.
The effect of Triton X-100 on the TPA-based buffer was much
smaller. Triton X-100 differs from the other detergents present in
that it contains a phenol ether moiety. Applicants hypothesize that
the beneficial effect of Triton X-100 may result from the oxidation
of the phenol ether moiety at the electrode and the participation
of the Triton oxidation product in the ECL reaction.
Surprisingly, the effect of detergents on assays conducted on
non-plasma treated plates was different and much greater in
magnitude. FIG. 7 shows the effect of five different non-ionic
detergents on TPA/Phos, TPA/Tris, TPA/Gly-Gly and PIPES/Phos Assay
Buffers (the optimized formulations of Example IV except for the
composition and amount of detergent). Tween 20, Thesit, Triton
X-100 and Triton X-114 were all present at a concentration of
0.05%(v/v). .beta.-Octyl glucopyranoside was present at a
concentration of 4 %(v/v). In this experiment, streptavidin-coated
electrodes were treated with 0.018 pmol of BT-IgG* (6.3 labels per
protein) in a volume of 50 .mu.L. The figure shows that all the
detergents significantly improved the ECL signal measured in the
presence of TPA-containing Assay Buffers relative to the same Assay
Buffer in the absence of detergent. In most cases, the improvement
was greater than 2 fold. In additional experiments, it was observed
that the maximal signals observed in each Assay Buffer tended to
occur at the critical micellar concentration (cmc) of a detergent
or higher. In contrast to the TPA-containing ECL Assay Buffers, the
performance of PIPES was improved .about.30 fold by the addition of
the phenol ether containing detergents (Triton X-100 and Triton
X-114) but very little improvement in signal was observed in the
presence of the Tween and Thesit detergents.
Applicants hypothesize that the effect of the Triton detergents on
the PIPES/Phos buffer may be related to the participation of Triton
oxidation products in the ECL process. By contrast, the effect of
detergents on the ECL signal from TPA-containing Assay Buffers on
NPT electrodes appears to be a more general phenomenon and may
relate to the stabilization of TPA oxidation products in detergent
micelles.
A larger screen of detergents was conducted to identify other
detergents that improved the ECL signal from BT-IgG* on
streptavidin-coated NPT electrodes in the presence of
TPA/Phosphate. The addition of all the non-ionic detergents
(APO-14, Triton X-100, .beta.-nonyl-glucoside, Tween 20, Genapol
and pentaethylene glycol mono-n-dodecyl ether) and zwitterionic
detergents (ASB-14 and Empigen) that were tested produced increases
in the ECL signal.
Example VII
ECL Activity of Selcted Tertiary Amines
A number of tertiary amines were screened for their ability act as
coreactants. The measurements were conducted in a similar fashion
as the methods described in Examples IV and V. ECL Assay Buffers
were prepared that contained the selected tertiary amine (200 mM),
phosphate buffer (200 mM), Triton X-100 (0.1 %) and that were
adjusted to pH 7.5. The signal from label attached to an electrode
was measured using the following procedure: i) a solution
containing 0.3 nM Bt-IgG* (IgG labeled with Sulfo-TAG and biotin)
in an ECL Assay Buffer was introduced into the wells of
streptavidin-coated 96-well NPT or PT plates; ii) the plates were
incubated for 2 h with shaking to allow the Bt-IgG* to bind the
surface; iii) the plates were washed four times and 150 .mu.L of
the ECL Assay Buffer was added and iv) the ECL from the label was
measured. The assay buffer background was measured by introducing
150 .mu.L of an ECL Assay Buffer into a streptavidin-coated plate
and measuring the ECL. The ECL signal from free ECL labels in
solution was measured by introducing a solution containing 10 nM
IgG* (IgG labeled with Sulfo-TAG but not biotin) into the wells of
streptavidin-coated 96-well NPT or PT plates and measuring the
ECL.
Tables IIa and IIb presents the results of the experiments on NPT
and PT plates, respectively. The results show that a variety of
tertiary amines were suitable for use as coreactants. In general,
the introduction of functionalization appeared to improve the ratio
of bound to free signals. Tertiary amines having N-substituted
morpholine core or, even more advantageously, a di-N-substituted
piperazine core (especially, PIPES, HEPES, POPSO, HEPPSO and EPPS)
appeared to be especially well suited for distinguishing bound vs.
free signals (especially on NPT surfaces). There was some
difference in the relative performances on PT and NPT plates, e.g.,
MOPS was found to perform particularly well on PT plates while
bis-Tris-Propane gave exceptionally high signals on NPT plates.
ECL was also measured using coreactants in comparable buffers,
except they did not include detergent or Tween 20 was used as the
detergent. Two coreactants other than TPA stood out as having very
low dependence on the presence or absence of Triton X-100
(N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid and TPA
dimer). These detergents have bound/free ratios than TPA and are
especially suitable for unwashed assays having detergent sensitive
components.
Additional experiments showed that these coreactants could be used
in ECL Assay buffers buffered with a variety of different pH
buffers, e.g., GlyGly, Gly, Tris, Tricine and phosphate.
TABLE-US-00003 TABLE IIa NPT Plates ECL Bound Bound Tertiary Amine
Background Free Bound Background Free
N-2-Hydroxypiperazine-N-2-ethanesulfonic acid (HEPES) 171 3716
73897 432 21 Piperazine-N,N'-bis-4-butanesulfonic acid 109 311
12490 115 61 Homopiperidine-N-3-propanesulfonic acid 921 13833
11683 13 1 Piperazine-N,N'-bis-3-propanesulfonic acid 92 362 12318
134 45 Piperidine-N-3-propanesulfonic acid 861 9417 16067 19 2
(3-[N-Morphilino)-3-propane sulfonic acid (MOPS) 177 658 5857 33 12
Piperazine-N-2-hydroxyethane-N'-3-methylpropanoate 128 340 12346 96
58 Piperazine-N,N'-bis-3-methylpropanoate 76 210 5377 71 40
1,6-diaminohexane-N,N,N',N'-tetraacetic acid 471 2522 29011 62 14
N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid 1446 5541 27561
19 6 N,N-bis propyl-N-4-aminobutanesulfonic acid 564 13795 49778 88
4 piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES) 163 777 41418
254 67 N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid
(TES) 282 804 16062 57 30
1,3-Bis[tris(hydroxymethyl)methylamino]propane (bis-Tris propane)
252 5207 61712 245 12 3-Dimethylamino-1-propanol 236 2946 18403 78
7 1-Dimethylamino-2-propanol 741 3463 22446 30 8
N,N,N',N'-tetrapropylpropane-1,3,-diamine 260 2397 36137 139 17 MSD
Assay Buffer (TPA) 490 15407 51137 104 3
Piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO) 283 1995
86494 306 50
2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic
acid 225 1482 81888 364 65 (HEPPSO)
3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS)
215 1545 79148 368 59 N,N-bis(2-hydroxyethyl)-2-aminoethane
sulfonic acid (BES) 57 97 2009 35 49
TABLE-US-00004 TABLE IIb PT Plates ECL Bound Bound Tertiary Amine
Background Free Bound Background Free
N-2-Hydroxypiperazine-N-2-ethanesulfonic acid (HEPES) 148 363 4233
29 19 Piperazine-N,N'-bis-4-butanesulfonic acid 111 152 750 7 16
Homopiperidine-N-3-propanesulfonic acid 447 4347 14130 32 4
Piperazine-N,N'-bis-3-propanesulfonic acid 88 120 499 6 13
Piperidine-N-3-propanesulfonic acid 376 2234 6148 16 3
(3-[N-Morphilino)-3-propane sulfonic acid (MOPS) 294 388 4221 14 42
Piperazine-N-2-hydroxyethane-N'-3-methylpropanoate 155 234 1624 10
19 Piperazine-N,N'-bis-3-methylpropanoate 182 287 1807 10 15
1,6-diaminohexane-N,N,N',N'-tetraacetic acid 389 1160 7898 20 10
N,N-bis-(hydroxyethyl)-N-4-aminobutanesulfonic acid 297 452 5560 19
34 N,N-bis propyl-N-4-aminobutanesulfonic acid 247 3685 16465 67 5
piperazine-N,N'-bis(2-ethane sulfonic acid) (PIPES) 297 452 5560 19
34 N-tris(hydroxymethyl)methyl-2-aminoethane sulfonic acid (TES)
324 455 3050 9 21 1,3-Bis[tris(hydroxymethyl)methylamino]propane
(bis-Tris propane) 181 382 2559 14 12 3-Dimethylamino-1-propanol
207 1079 5414 26 6 1-Dimethylamino-2-propanol 545 1332 6903 13 8
N,N,N',N'-tetrapropylpropane-1,3,-diamine 237 983 9468 40 12 MSD
Assay Buffer (TPA) 295 7162 18915 64 3
Piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO) 338 657
3842 11 11
2-hydroxy-3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic
acid 142 207 749 5 9 (HEPPSO)
3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1-sulfonic acid (EPPS)
132 207 803 6 9 N,N-bis(2-hydroxyethyl)-2-aminoethane sulfonic acid
(BES) 112 118 636 6 87
7. INCORPORATION OF REFERENCES
The present invention is not to be limited in scope by the specific
embodiments described herein. Indeed, various modifications of the
invention in addition to those described herein will become
apparent to those skilled in the art from the foregoing description
and accompanying figures. Such modifications are intended to fall
within the scope of the claims. Various publications are cited
herein, the disclosures of which are incorporated by reference in
their entireties.
* * * * *